Antagonist anti-notch3 antibodies and their use in the prevention and treatment of notch3-related diseases

ABSTRACT

The present invention relates to antagonist antibodies that specifically bind to Notch 3 and inhibit its activation. The present invention includes antibodies binding to a conformational epitope comprising the first Lin12 domain and the second dimerization domain. The present invention also includes uses of these antibodies to treat or prevent Notch 3 related diseases or disorders.

RELATED APPLICATIONS

This application is a divisional application of U.S. patent applicationSer. No. 11/958,099, filed Dec. 17, 2007, which claims the benefit ofU.S. Provisional Patent Application No. 60/875,597, filed Dec. 18, 2006,and U.S. Provisional Patent Application No. 60/879,218, filed Jan. 6,2007. The disclosures of the foregoing applications are incorporatedherein by reference in their entirety.

FIELD OF THE INVENTION

The present invention relates to antagonist anti-Notch3 antibodies andtheir use in the amelioration, treatment, or prevention of aNotch3-related disease or disorder.

BACKGROUND OF THE INVENTION

The Notch gene was first described in 1917 when a strain of the fruitfly Drosophila melanogaster was found to have notched wing blades(Morgan, Am Nat 51:513 (1917)). The gene was cloned almost seventy yearslater and was determined to be a cell surface receptor playing a keyrole in the development of many different cell types and tissues inDrosophila (Wharton et al., Cell 43:567 (1985)). The Notch signalingpathway was soon found to be a signaling mechanism mediated by cell-cellcontact and has been evolutionarily conserved from Drosophila to human.Notch receptors have been found to be involved in many cellularprocesses, such as differentiation, cell fate decisions, maintenance ofstem cells, cell motility, proliferation, and apoptosis in various celltypes during development and tissue homeostasis (For review, seeArtavanis-Tsakonas, et al., Science 268:225 (1995)).

Mammals possess four Notch receptor proteins (designated Notch1 toNotch4) and five corresponding ligands (designated Delta-1 (DLL-1),Delta-3 (DLL-3), Delta-4 (DLL-4), Jagged-1 and Jagged-2). The mammalianNotch receptor genes encode ˜300 kD proteins that are cleaved duringtheir transport to the cell surface and exist as heterodimers. Theextracellular portion of the Notch receptor has thirty-four epidermalgrowth factor (EGF)-like repeats and three cysteine-rich Notch/LIN12repeats. The association of two cleaved subunits is mediated bysequences lying immediately N-terminal and C-terminal of the cleavagesite, and these two subunits constitute the Notch heterodimerization(HD) domains (Wharton, et al., Cell 43:567 (1985); Kidd, et al., MolCell Biol 6:3431 (1986); Kopczynski, et al., Genes Dev 2:1723 (1988);Yochem, et al., Nature 335:547 (1988)).

At present, it is still not clear how Notch signaling is regulated bydifferent receptors or how the five ligands differ in their signaling orregulation. The differences in signaling and/or regulation may becontrolled by their expression patterns in different tissues or bydifferent environmental cues. It has been documented that Notch ligandproteins, including Jagged/Serrate and Delta/Delta-like, specificallybind to the EGF repeat region and induce receptor-mediated Notchsignaling (reviewed by Bray, Nature Rev Mol Cell Biol. 7:678 (2006), andby Kadesch, Exp Cell Res. 260:1 (2000)). Among the EGF repeats, the 10thto 12th repeats are required for ligand binding to the Notch receptor,and the other EGF repeats may enhance receptor-ligand interaction (Xu,et al., J Biol Chem. 280:30158 (2005); Shimizu, et al., Biochem BiophysRes Comm. 276:385 (2000)). Although the LIN12 repeats and thedimerization domain are not directly involved in ligand binding, theyplay important roles in maintaining the heterodimeric protein complex,preventing ligand-independent protease cleavage and receptor activation(Sanche-Irizarry, et al., Mol Cell Biol. 24:9265 (2004); Vardar et al.,Biochem. 42:7061 (2003)).

The expression of mutant forms of Notch receptors in developing Xenopusembryos interferes profoundly with normal development (Coffman, et al.,Cell 73: 659 (1993)). A Notch1 knockout was found to be embryonic lethalin mice (Swiatek, et al., Genes & Dev 8:707 (1994)). In humans, therehave been several genetic diseases, including cancer, linked todifferent Notch receptor mutations (Artavanis-Tsakonas, et al., Science284:770 (1999)). For instance, aberrant activation of Notch1 receptorcaused by translocation can lead to T cell lymphoblastic leukemia(Ellisen, et al., Cell 66:649 (1991)). Certain mutations in the HDdomains of Notch1 receptor enhance signaling without ligand binding(Malecki, et al., Mol Cell Biol 26:4642 (2006)), further implicatingtheir roles in Notch receptor activation. The signal induced by ligandbinding is transmitted to the nucleus by a process involving twoproteolytic cleavages of the receptor followed by nuclear translocationof the intracellular domain (Notch-IC). Although LIN12 repeats and HDdomains were thought to prevent signaling in the absence of ligands, itis still unclear how ligand binding facilitates proteolytic cleavageevents.

Notch receptors have been linked to a wide range of diseases includingcancer, neurological disorders, and immune diseases, as evidenced byreports of the over-expression of Notch receptors in various humandisease tissues and cell lines as compared to normal or nonmalignantcells (Joutel, et al. Cell & Dev Biol 9:619 (1998); Nam, et al., CurrOpin Chem Biol 6:501 (2002)). The Notch3 receptor is over-expressed invarious solid tumors, including non-small cell lung cancer (NSCLC) andovarian cancer (Haruki, et al., Cancer Res 65:3555 (2005); Park, et al.,Cancer Res 66:6312 (2006); Lu, et al., Clin Cancer Res 10:3291 (2004)),suggesting the significance of Notch3 receptor expression in solidtumors. Furthermore, Notch3 receptor expression is upregulated in plasmacell neoplasms, including multiple myeloma, plasma cell leukemia, andextramedullary plasmacytoma (Hedvat, et al., Br J Haematol 122:728(2003); pancreatic cancer (Buchler, et al., Ann Surg 242:791 (2005));and T cell acute lymphoblastic leukemias (T-ALL) (Bellavia, et al., ProcNatl Acad Sci USA 99:3788 (2002); Screpanti, et al., Trends Mol Med 9:30(2003)). Notch3 receptor is also expressed in a subset of neuroblastomacell lines and serves as a marker for this type of tumor that hasconstitutional or tumor-specific mutations in the homeobox gene Phox2B(van Limpt, et al., Cancer Lett 228:59 (2005)). Other indications anddiseases that have been linked to Notch3 receptor expression includeneurological disorders (Joutel, et al., Nature 383:707 (1996)), diabetes(Anastasi, et al., J Immunol 171:4504 (2003), rheumatoid arthritis(Yabe, et al., J Orthop Sci 10:589 (2005)), vascular related diseases(Sweeney, et al., FASEB J 18:1421 (2004)), and Alagille syndrome (Flynn,et al., J Pathol 204:55 (2004)).

Although Notch3 receptor over-expression (including gene amplification)has been observed in various cancers, no activating mutations have yetbeen reported. It is plausible that an increased level of Notch3receptors in tumors can be activated by different ligands in stromalcells or tumor cells and lead to enhanced Notch3 signaling.Particularly, Notch ligands have been localized to the vascularendothelium during both development and tumorigenesis (Mailhos, et al.,Differentiation 69:135 (2001); Taichman, et al., Dev Dyn 225: 166(2002)), suggesting endothelial cells could provide the ligands forNotch3 receptor activation in tumors. Similar tumor-stroma cross-talkmediated by Notch ligand and receptor have been demonstrated indifferent type of cancers (Houde, et al., Blood 104: 3697 (2004); Jundt,et al., Blood 103: 3511 (2004); Zeng, et al., Cancer Cell 8: 13 (2005)).Increased Notch3 signaling caused by over-expression of intracellularNotch3 (Notch3-IC) can lead to tumorigenesis in T-ALL and breast canceranimal models (Vacca, et al., The EMBO J 25: 1000 (2006); Hu, et al., AmJ Pathol 168: 973 (2006)).

Notch signaling and its role in cell self-renewal have been implicatedin cancer stem cells, which are a minority population in tumors and caninitiate tumor formation (Reya, et al., Nature 414:105 (2001)). Normalstem cells from many tissues, including intestinal and neuronal stemcells, depend on Notch signaling for self-renewal and fate determination(Fre, et al., Nature, 435: 964 (2005); van Es, et al., Nature, 435:959(2005); Androutsellis-Theotokis, et al., Nature, 442: 823 (2006)).Similar mechanisms could exist in cancer stem cells, and inhibition ofNotch signaling by γ-secretase inhibitors was shown to deplete cancerstem cells and block engraftment in embryonal brain tumors (Fan, et al.,Cancer Res 66:7445 (2006)).

Inhibition of Notch signaling by γ-secretase inhibitor has strikingantineoplastic effects in Notch-expressing transformed cells in vitroand in xenograft models (Weijzen, et al., Nat Medicine 8: 879 (2002);Bocchetta, et al., Oncogene 22:81 (2003); Weng, et al., Science, 306:269(2004)). More recently, a γ-secretase inhibitor has been shown toefficaciously kill colon adenomas in Apc (min+) mice (van Es, et al.,Nature, 435: 959 (2005)), although the therapeutic window, due to itseffect on normal stem cells and the inhibition of multiple Notchpathways, is very narrow. Different from Notch1, a Notch3 gene knockoutin mice was not embryonically lethal and had few defects (Domenga, etal., Genes & Dev 18: 2730 (2004)), suggesting that Notch 3 provides apotentially better therapeutic target than Notch 1.

Tournier-Lasserve et al. (U.S. Application 2003/0186290) teach theassociation of Notch3 receptor and CADASIL. The application disclosesvarious mutations in the Notch3 gene and their possible association withthe disease CADASIL. The application suggests the use of diagnosticantibodies to detect such mutations. The application also suggeststherapeutic antibodies to treat CADASIL, i.e. agonistic antibodies, butno specific antibodies are disclosed nor how to make such antibodies.

In view of the large number of human diseases associated with the Notch3signaling pathway, it is important that new ways of preventing andtreating these diseases be identified. The current invention providesnovel anti-Notch3 antibodies useful for this unmet medical need.

SUMMARY OF THE INVENTION

The present invention provides novel antibodies and fragments thereofthat specifically bind to a conformational epitope of the human Notch3receptor, the epitope comprising the LIN12 domain and theheterodimerization domain. Another aspect of the invention includes theepitope binding site and antibodies that bind this same epitope as theantibodies of the present invention. The antibodies of the presentinvention inhibit ligand-induced signaling through the Notch3 receptor.

The invention includes the amino acid sequences of the variable heavyand light chain of the antibodies and their corresponding nucleic acidsequences. Another embodiment of the invention includes the CDRsequences of these antibodies. Another embodiment includes humanizedforms of these antibodies.

Another embodiment of the present invention includes the cell lines andvectors harboring the antibody sequences of the present invention.

The present invention also includes the conformational epitoperecognized by the antagonist antibodies of the invention. The presentinvention also includes antibodies that bind this conformationalepitope. The embodiments include a Notch 3 conformational epitopecomprising the LIN12 domain having at least 80%, 85%, 90%, or 95%sequence identity with SEQ ID NO. 9 and the dimerization domain 2 havingat least 80%, 85%, 90%, or 95% sequence identity with SEQ ID NO. 18.More particularly, the Notch 3 conformational epitope comprising aminoacid residues 1395-1396, 1402-1404 and 1420-1422 of the L1 LIN12 domainand amino acid residues 1576-1578 and 1626-1628 of the D2 dimerizationdomain. The present invention includes antibodies that bind thisconformational epitope.

Another embodiment of the preset invention is the use of any of theseantibodies for the preparation of a medicament or composition for thetreatment of diseases and disorders associated with Notch 3 receptoractivation.

Another embodiment of the preset invention is the use of any of theseantibodies in the treatment of disorders associated with Notch 3activation comprising the inhibition of said activation by, e.g.,inhibiting Notch 3 signaling, or neutralization of the receptor byblocking ligand binding. Notch 3 related disorders may include, but arenot limited to, T-cell acute lymphoblastic leukemia, lymphoma, liverdisease involving aberrant vascularization, diabetes, ovarian cancer,diseases involving vascular cell fate, rheumatoid arthritis, pancreaticcancer, non-small cell lung cancer, plasma cell neoplasms (such asmultiple myeloma, plasma cell leukemia, and extramedullaryplasmacytoma), and neuroblastoma.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 depicts the amino acid sequence of Notch3. The EGF repeat regionextends from amino acid residue 43 to 1383; the LIN12 domain extendsfrom amino acid residue 1384 to 1503; and the dimerization domainextends from amino acid residue 1504 to 1640.

FIG. 2 (A-H) depicts the amino acid sequence comparison between humanNotch 1 (SEQ ID NO:44), Notch 2 (SEQ ID NO:45), Notch 3 (SEQ ID NO:1),and Notch 4 (SEQ ID NO:46).

FIG. 3 depicts the percent identity of Notch 1, Notch 2, Notch 3, andNotch 4.

FIGS. 4A and 4B depict the heavy and light chain variable regionsequences of anti-Notch3 monoclonal antibody MAb 256A-4 (SEQ ID NO:2 andSEQ ID NO:3, respectively), with CDR regions underlined.

FIGS. 5A and 5B depict the heavy and light chain variable regionsequences of anti-Notch3 monoclonal antibody MAb 256A-8 (SEQ ID NO: 4and SEQ ID NO:5, respectively), with CDR regions underlined.

FIG. 6 depicts a luciferase reporter assay of Example 5 showinginhibitory effects by anti-Notch3 MAbs on the Notch3 ligand Jagged 1.

FIG. 7 depicts the luciferase reporter assay showing inhibitory effectsby anti-Notch3 MAbs on the Notch3 ligand Jagged 2.

FIG. 8 depicts the luciferase reporter assay showing inhibitory effectsby anti-Notch3 MAbs on the Notch3 ligand DLL4.

FIG. 9 depicts the luciferase reporter assay showing inhibitory effectsto native Notch3 in ovarian cancer cells by anti-Notch3 MAbs. (9A) Humanovarian cancer cell line, OV/CAR3 and (9B) Human ovarian cancer cellline, A2780.

FIG. 10 depicts the apoptosis assay of Example 6 showing that cellsurvival effect induced by Jagged1 was inhibited by anti-Notch3 MAbs.

FIG. 11 depicts the inhibitory effect of anti-Notch3 MAbs on cellmigration (11A) and invasion (11B) of Example 7.

FIG. 12 depicts a schematic diagram of the Notch1-Notch3 domain-swapprotein expressed as a fusion protein with human IgG/Fc linked toC-terminus.

FIG. 13A depicts an ELISA using anti-human Fc control antibody as thedetection antibody showing that the proteins of FIG. 12 were expressedin conditioned medium. FIG. 13B depicts an ELISA using 256A-4 as thedetection antibody. FIG. 13C depicts an ELISA using 256A-8 as thedetection antibody. FIG. 13D depicts an ELISA using a positive controlantibody 256A-13 as the detection antibody.

FIG. 14 depicts the comparison of the engineered Notch3 leader peptidecoding sequence (SEQ ID NO:47) to the native Notch3 leader peptidecoding sequence (SEQ ID NO:48) (NCBI GENBANK® Accession No.NM_(—)000435) showing the changes of nucleotides (14A) and thetranslated amino acid sequence of the engineered Notch leader peptidesequence (SEQ ID NO:6) (14B).

FIG. 15 depicts the generation of domain swap construct by PCR-SOEmethod. Arrow bars represent PCR primers. Open bar, Notch3 sequence.Filled bar, Notch1 sequence.

FIG. 16 depicts the amino acid sequences used in the Notch3 LIN12 domainepitope mapping of the MAb 256A-4 and 256A-8.

FIG. 17 depicts the amino acid sequences used in the Notch3 dimerizationdomain epitope mapping of the MAb 256A-4 and 256A-8.

FIG. 18 depicts a schematic of the epitope binding site for MAb 256A-4and 256A-8.

DETAILED DESCRIPTION

This invention is not limited to the particular methodology, protocols,cell lines, vectors, or reagents described herein because they may vary.Further, the terminology used herein is for the purpose of describingparticular embodiments only and is not intended to limit the scope ofthe present invention. As used herein and in the appended claims, thesingular forms “a”, “an”, and “the” include plural reference unless thecontext clearly dictates otherwise, e.g., reference to “a host cell”includes a plurality of such host cells. Unless defined otherwise, alltechnical and scientific terms and any acronyms used herein have thesame meanings as commonly understood by one of ordinary skill in the artin the field of the invention. Although any methods and materialssimilar or equivalent to those described herein can be used in thepractice of the present invention, the exemplary methods, devices, andmaterials are described herein.

All patents and publications mentioned herein are incorporated herein byreference to the extent allowed by law for the purpose of describing anddisclosing the proteins, enzymes, vectors, host cells, and methodologiesreported therein that might be used with the present invention. However,nothing herein is to be construed as an admission that the invention isnot entitled to antedate such disclosure by virtue of prior invention.

Definitions

Terms used throughout this application are to be construed with ordinaryand typical meaning to those of ordinary skill in the art. However,Applicants desire that the following terms be given the particulardefinitions as defined below.

The phrase “substantially identical” with respect to an antibody chainpolypeptide sequence may be construed as an antibody chain exhibiting atleast 70%, or 80%, or 90%, or 95% sequence identity to the referencepolypeptide sequence. The term with respect to a nucleic acid sequencemay be construed as a sequence of nucleotides exhibiting at least about85%, or 90%, or 95%, or 97% sequence identity to the reference nucleicacid sequence.

The term “identity” or “homology” shall be construed to mean thepercentage of amino acid residues in the candidate sequence that areidentical with the residue of a corresponding sequence to which it iscompared, after aligning the sequences and introducing gaps, ifnecessary to achieve the maximum percent identity for the entiresequence, and not considering any conservative substitutions as part ofthe sequence identity. Neither N- nor C-terminal extensions norinsertions shall be construed as reducing identity or homology. Methodsand computer programs for the alignment are well known in the art.Sequence identity may be measured using sequence analysis software.

The term “antibody” is used in the broadest sense, and specificallycovers monoclonal antibodies (including full length monoclonalantibodies), polyclonal antibodies, and multispecific antibodies (e.g.,bispecific antibodies), and antibody fragments so long as they exhibitthe desired biological activity. Antibodies (Abs) and immunoglobulins(Igs) are glycoproteins having the same structural characteristics.While antibodies exhibit binding specificity to a specific target,immunoglobulins include both antibodies and other antibody-likemolecules which lack target specificity. The antibodies of the inventioncan be of any type (e.g., IgG, IgE, IgM, IgD, IgA and IgY), class (e.g.,IgG1, IgG2, IgG3, IgG4, IgA1 and IgA2) or subclass. Native antibodiesand immunoglobulins are usually heterotetrameric glycoproteins of about150,000 Daltons, composed of two identical light (L) chains and twoidentical heavy (H) chains. Each heavy chain has at one end a variabledomain (V_(H)) followed by a number of constant domains. Each lightchain has a variable domain at one end (V_(L)) and a constant domain atits other end.

As used herein, “anti-Notch3 antibody” means an antibody which bindsspecifically to human Notch3 in such a manner so as to inhibit orsubstantially reduce the binding of Notch3 to its ligands or to inhibitNotch 3 signaling.

The term “variable” in the context of variable domain of antibodies,refers to the fact that certain portions of the variable domains differextensively in sequence among antibodies and are used in the binding andspecificity of each particular antibody for its particular target.However, the variability is not evenly distributed through the variabledomains of antibodies. It is concentrated in three segments calledcomplementarity determining regions (CDRs; i.e., CDR1, CDR2, and CDR3)also known as hypervariable regions both in the light chain and theheavy chain variable domains. The more highly conserved portions ofvariable domains are called the framework (FR). The variable domains ofnative heavy and light chains each comprise four FR regions, largely aadopting a β-sheet configuration, connected by three CDRs, which formloops connecting, and in some cases forming part of, the β-sheetstructure. The CDRs in each chain are held together in close proximityby the FR regions and, with the CDRs from the other chain, contribute tothe formation of the target binding site of antibodies (see Kabat, etal. Sequences of Proteins of Immunological Interest, National Instituteof Health, Bethesda, Md. (1987)). As used herein, numbering ofimmunoglobulin amino acid residues is done according to theimmunoglobulin amino acid residue numbering system of Kabat, et al.,unless otherwise indicated.

The term “antibody fragment” refers to a portion of a full-lengthantibody, generally the target binding or variable region. Examples ofantibody fragments include F(ab), F(ab′), F(ab′)₂ and Fv fragments. Thephrase “functional fragment or analog” of an antibody is a compoundhaving qualitative biological activity in common with a full-lengthantibody. For example, a functional fragment or analog of an anti-Notch3antibody is one which can bind to a Notch3 receptor in such a manner soas to prevent or substantially reduce the ability of the receptor tobind to its ligands or initiate signaling. As used herein, “functionalfragment” with respect to antibodies, refers to Fv, F(ab) and F(ab′)₂fragments. An “Fv” fragment consists of a dimer of one heavy and onelight chain variable domain in a tight, non-covalent association(V_(H)-V_(L) dimer). It is in this configuration that the three CDRs ofeach variable domain interact to define a target binding site on thesurface of the V_(H)-V_(L) dimer. Collectively, the six CDRs confertarget binding specificity to the antibody. However, even a singlevariable domain (or half of an Fv comprising only three CDRs specificfor a target) has the ability to recognize and bind target, although ata lower affinity than the entire binding site.

“Single-chain Fv” or “sFv” antibody fragments comprise the V_(H) andV_(L) domains of an antibody, wherein these domains are present in asingle polypeptide chain. Generally, the Fv polypeptide furthercomprises a polypeptide linker between the V_(H) and V_(L) domains whichenables the sFv to form the desired structure for target binding.

The term “diabodies” refers to small antibody fragments with twoantigen-binding sites, which fragments comprise a heavy chain variabledomain (V_(H)) connected to a light chain variable domain (V_(L)) in thesame polypeptide chain. By using a linker that is too short to allowpairing between the two domains on the same chain, the domains areforced to pair with the complementary domains of another changing andcreate two antigen-binding sites.

The F(ab) fragment contains the constant domain of the light chain andthe first constant domain (CH1) of the heavy chain. F(ab′) fragmentsdiffer from F(ab) fragments by the addition of a few residues at thecarboxyl terminus of the heavy chain CH1 domain including one or morecysteines from the antibody hinge region. F(ab′) fragments are producedby cleavage of the disulfide bond at the hinge cysteines of the F(ab′)₂pepsin digestion product. Additional chemical couplings of antibodyfragments are known to those of ordinary skill in the art.

The term “monoclonal antibody” as used herein refers to an antibodyobtained from a population of substantially homogeneous antibodies,i.e., the individual antibodies comprising the population are identicalexcept for possible naturally occurring mutations that may be present inminor amounts. Monoclonal antibodies herein specifically include“chimeric” antibodies (immunoglobulins) in which a portion of the heavyand/or light chain is identical with or homologous to correspondingsequences in antibodies derived from a particular species or belongingto a particular antibody class or subclass, while the remainder of thechain(s) is identical with or homologous to corresponding sequences inantibodies derived from another species or belonging to another antibodyclass or subclass, as well as fragments of such antibodies, so long asthey exhibit the desired biological activity (U.S. Pat. No. 4,816,567;and Morrison, et al., Proc Natl Acad Sci USA 81:6851 (1984)). Monoclonalantibodies are highly specific, being directed against a single targetsite. Furthermore, in contrast to conventional (polyclonal) antibodypreparations which typically include different antibodies directedagainst different determinants (epitopes), each monoclonal antibody isdirected against a single determinant on the target. In addition totheir specificity, monoclonal antibodies are advantageous in that theymay be synthesized by the hybridoma culture, uncontaminated by otherimmunoglobulins. The modifier “monoclonal” indicates the character ofthe antibody as being obtained from a substantially homogeneouspopulation of antibodies, and is not to be construed as requiringproduction of the antibody by any particular method. For example, themonoclonal antibodies for use with the present invention may be isolatedfrom phage antibody libraries using well known techniques. The parentmonoclonal antibodies to be used in accordance with the presentinvention may be made by the hybridoma method first described by Kohler,et al., Nature 256:495 (1975), or may be made by recombinant methods.

“Humanized” forms of non-human (e.g., murine) antibodies are chimericimmunoglobulins, immunoglobulin chains or fragments thereof (such as Fv,Fab, Fab′, F(ab′)₂ or other target-binding subsequences of antibodies)which contain minimal sequence derived from non-human immunoglobulin. Ingeneral, the humanized antibody will comprise substantially all of atleast one, and typically two, variable domains, in which all orsubstantially all of the CDR regions correspond to those of a non-humanimmunoglobulin and all or substantially all of the FR regions are thoseof a human immunoglobulin template sequence. The humanized antibody mayalso comprise at least a portion of an immunoglobulin constant region(Fc), typically that of a human immunoglobulin template chosen.

The terms “cell,” “cell line,” and “cell culture” include progeny. It isalso understood that all progeny may not be precisely identical in DNAcontent, due to deliberate or inadvertent mutations. Variant progenythat have the same function or biological property, as screened for inthe originally transformed cell, are included. The “host cells” used inthe present invention generally are prokaryotic or eukaryotic hosts.

“Transformation” of a cellular organism, cell, or cell line with DNAmeans introducing DNA into the target cell so that the DNA isreplicable, either as an extrachromosomal element or by chromosomalintegration. “Transfection” of a cell or organism with DNA refers to thetaking up of DNA, e.g., an expression vector, by the cell or organismwhether or not any coding sequences are in fact expressed. The terms“transfected host cell” and “transformed” refer to a cell in which DNAwas introduced. The cell is termed “host cell” and it may be eitherprokaryotic or eukaryotic. Typical prokaryotic host cells includevarious strains of E. coli. Typical eukaryotic host cells are mammalian,such as Chinese hamster ovary or cells of human origin. The introducedDNA sequence may be from the same species as the host cell or adifferent species from the host cell, or it may be a hybrid DNAsequence, containing some foreign and some homologous DNA.

The term “vector” means a DNA construct containing a DNA sequence whichis operably linked to a suitable control sequence capable of effectingthe expression of the DNA sequence in a suitable host. Such controlsequences include a promoter to effect transcription, an optionaloperator sequence to control such transcription, a sequence encodingsuitable mRNA ribosome binding sites, and sequences which control thetermination of transcription and translation. The vector may be aplasmid, a phage particle, or simply a potential genomic insert. Oncetransformed into a suitable host, the vector may replicate and functionindependently of the host genome, or may in some instances, integrateinto the genome itself. In the present specification, “plasmid” and“vector” are sometimes used interchangeably, as the plasmid is the mostcommonly used form of vector. However, the invention is intended toinclude such other forms of vectors which serve equivalent function asand which are, or become, known in the art.

“Mammal” for purposes of treatment refers to any animal classified as amammal, including human, domestic and farm animals, nonhuman primates,and zoo, sports, or pet animals, such as dogs, horses, cats, cows, etc.

The word “label” when used herein refers to a detectable compound orcomposition which can be conjugated directly or indirectly to a moleculeor protein, e.g., an antibody. The label may itself be detectable (e.g.,radioisotope labels or fluorescent labels) or, in the case of anenzymatic label, may catalyze chemical alteration of a substratecompound or composition which is detectable.

As used herein, “solid phase” means a non-aqueous matrix to which theantibody of the present invention can adhere. Examples of solid phasesencompassed herein include those formed partially or entirely of glass(e.g., controlled pore glass), polysaccharides (e.g., agarose),polyacrylamides, polystyrene, polyvinyl alcohol, and silicones. Incertain embodiments, depending on the context, the solid phase cancomprise the well of an assay plate; in others it is a purificationcolumn (e.g., an affinity chromatography column).

As used herein, the term “Notch3-mediated disorder” means a condition ordisease which is characterized by the overexpression and/orhypersensitivity of the Notch3 receptor. Specifically it would beconstrued to include conditions associated with cancers such asnon-small cell lung cancer, ovarian cancer, and T-cell acutelymphoblastic leukemia. Other cancers including pancreatic, prostatecancer, plasma cell neoplasms (e.g., multiple myeloma, plasma cellleukemia and extramedullary plasmacytoma), neuroblastoma andextramedullary plasmacytoma are also encompassed under the scope of thisterm. Other types of diseases include lymphoma, Alagille syndrome, liverdisease involving aberrant vascularization, neurologic diseases,diabetes, diseases involving vascular cell fate, and rheumatoidarthritis.

Notch 3 Receptor Immunogen for Generating Antibodies

Soluble targets or fragments thereof can be used as immunogens forgenerating antibodies. The antibody is directed against the target ofinterest. Preferably, the target is a biologically important polypeptideand administration of the antibody to a mammal suffering from a diseaseor disorder can result in a therapeutic benefit in that mammal. Wholecells may be used as the immunogen for making antibodies. The immunogenmay be produced recombinantly or made using synthetic methods. Theimmunogen may also be isolated from a natural source.

For transmembrane molecules, such as receptors, fragments of these(e.g., the extracellular domain of a receptor) can be used as theimmunogen. Alternatively, cells expressing the transmembrane moleculecan be used as the immunogen. Such cells can be derived from a naturalsource (e.g., cancer cell lines) or may be cells which have beentransformed by recombinant techniques to over-express the transmembranemolecule. Other forms of the immunogen useful for preparing antibodieswill be apparent to those in the art.

Alternatively, a gene or a cDNA encoding human Notch3 receptor may becloned into a plasmid or other expression vector and expressed in any ofa number of expression systems according to methods well known to thoseof skill in the art. Methods of cloning and expressing Notch3 receptorand the nucleic acid sequence for human Notch3 receptor are known (see,for example, U.S. Pat. Nos. 5,821,332 and 5,759,546). Because of thedegeneracy of the genetic code, a multitude of nucleotide sequencesencoding Notch3 receptor protein or polypeptides may be used. One mayvary the nucleotide sequence by selecting combinations based on possiblecodon choices. These combinations are made in accordance with thestandard triplet genetic code as applied to the nucleotide sequence thatcodes for naturally occurring Notch3 receptor and all such variationsmay be considered. Any one of these polypeptides may be used in theimmunization of an animal to generate antibodies that bind to humanNotch3 receptor.

Recombinant Notch3 proteins from other species may also be used asimmunogen to generate antibodies because of the high degree ofconservation of the amino acid sequence of Notch3. A comparison betweenhuman and mouse Notch3 showed over 90% amino acid sequence identitybetween the two species.

The immunogen Notch3 receptor may, when beneficial, be expressed as afusion protein that has the Notch3 receptor attached to a fusionsegment. The fusion segment often aids in protein purification, e.g., bypermitting the fusion protein to be isolated and purified by affinitychromatography, but can also be used to increase immunogenicity. Fusionproteins can be produced by culturing a recombinant cell transformedwith a fusion nucleic acid sequence that encodes a protein including thefusion segment attached to either the carboxyl and/or amino terminal endof the protein. Fusion segments may include, but are not limited to,immunoglobulin Fc regions, glutathione-S-transferase, β-galactosidase, apoly-histidine segment capable of binding to a divalent metal ion, andmaltose binding protein.

Recombinant Notch3 receptor protein as described in Example 1 was usedto immunize mice to generate the hybridomas that produce the monoclonalantibodies of the present invention. Exemplary polypeptides comprise allor a portion of SEQ ID NO. 1 or variants thereof.

Antibody Generation

The antibodies of the present invention may be generated by any suitablemethod known in the art. The antibodies of the present invention maycomprise polyclonal antibodies. Methods of preparing polyclonalantibodies are known to the skilled artisan (Harlow, et al., Antibodies:a Laboratory Manual, Cold spring Harbor Laboratory Press, 2nd ed.(1988), which is hereby incorporated herein by reference in itsentirety).

For example, an immunogen as described in Example 1 may be administeredto various host animals including, but not limited to, rabbits, mice,rats, etc., to induce the production of sera containing polyclonalantibodies specific for the antigen. The administration of the immunogenmay entail one or more injections of an immunizing agent and, ifdesired, an adjuvant. Various adjuvants may be used to increase theimmunological response, depending on the host species, and include butare not limited to, Freund's (complete and incomplete), mineral gelssuch as aluminum hydroxide, surface active substances such aslysolecithin, pluronic polyols, polyanions, peptides, oil emulsions,keyhole limpet hemocyanins, dinitrophenol, and potentially useful humanadjuvants such as BCG (bacille Calmette-Guerin) and Corynebacteriumparvum. Additional examples of adjuvants which may be employed includethe MPL-TDM adjuvant (monophosphoryl lipid A, synthetic trehalosedicorynomycolate). Immunization protocols are well known in the art andmay be performed by any method that elicits an immune response in theanimal host chosen. Adjuvants are also well known in the art.

Typically, the immunogen (with or without adjuvant) is injected into themammal by multiple subcutaneous or intraperitoneal injections, orintramuscularly or through IV. The immunogen may include a Notch3polypeptide, a fusion protein, or variants thereof. Depending upon thenature of the polypeptides (i.e., percent hydrophobicity, percenthydrophilicity, stability, net charge, isoelectric point etc.), it maybe useful to conjugate the immunogen to a protein known to beimmunogenic in the mammal being immunized. Such conjugation includeseither chemical conjugation by derivatizing active chemical functionalgroups to both the immunogen and the immunogenic protein to beconjugated such that a covalent bond is formed, or throughfusion-protein based methodology, or other methods known to the skilledartisan. Examples of such immunogenic proteins include, but are notlimited to, keyhole limpet hemocyanin, ovalbumin, serum albumin, bovinethyroglobulin, soybean trypsin inhibitor, and promiscuous T helperpeptides. Various adjuvants may be used to increase the immunologicalresponse as described above.

The antibodies of the present invention comprise monoclonal antibodies.Monoclonal antibodies are antibodies which recognize a single antigenicsite. Their uniform specificity makes monoclonal antibodies much moreuseful than polyclonal antibodies, which usually contain antibodies thatrecognize a variety of different antigenic sites. Monoclonal antibodiesmay be prepared using hybridoma technology, such as those described byKohler, et al., Nature 256:495 (1975); U.S. Pat. No. 4,376,110; Harlow,et al., Antibodies: A Laboratory Manual, Cold spring Harbor LaboratoryPress, 2nd ed. (1988) and Hammerling, et al., Monoclonal Antibodies andT-Cell Hybridomas, Elsevier (1981), recombinant DNA methods, or othermethods known to the artisan. Other examples of methods which may beemployed for producing monoclonal antibodies include, but are notlimited to, the human B-cell hybridoma technique (Kosbor, et al.,Immunology Today 4:72 (1983); Cole, et al., Proc Natl Acad Sci USA80:2026 (1983)), and the EBV-hybridoma technique (Cole, et al.,Monoclonal Antibodies and Cancer Therapy, pp. 77-96, Alan R. Liss(1985)). Such antibodies may be of any immunoglobulin class includingIgG, IgM, IgE, IgA, IgD and any subclass thereof. The hybridomaproducing the mAb of this invention may be cultivated in vitro or invivo.

In the hybridoma model, a host such as a mouse, a humanized mouse, amouse with a human immune system, hamster, rabbit, camel, or any otherappropriate host animal, is immunized to elicit lymphocytes that produceor are capable of producing antibodies that will specifically bind tothe protein used for immunization. Alternatively, lymphocytes may beimmunized in vitro. Lymphocytes then are fused with myeloma cells usinga suitable fusing agent, such as polyethylene glycol, to form ahybridoma cell (Goding, Monoclonal Antibodies: Principles and Practice,Academic Press, pp. 59-103 (1986)).

Generally, in making antibody-producing hybridomas, either peripheralblood lymphocytes (“PBLs”) are used if cells of human origin aredesired, or spleen cells or lymph node cells are used if non-humanmammalian sources are desired. The lymphocytes are then fused with animmortalized cell line using a suitable fusing agent, such aspolyethylene glycol, to form a hybridoma cell (Goding, MonoclonalAntibodies: Principles and Practice, Academic Press, pp. 59-103 (1986)).Immortalized cell lines are usually transformed mammalian cells,particularly myeloma cells of rodent, bovine or human origin. Typically,a rat or mouse myeloma cell line is employed. The hybridoma cells may becultured in a suitable culture medium that preferably contains one ormore substances that inhibit the growth or survival of the unfused,immortalized cells. For example, if the parental cells lack the enzymehypoxanthine guanine phosphoribosyl transferase (HGPRT or HPRT), theculture medium for the hybridomas typically will include hypoxanthine,aminopterin, and thymidine (“HAT medium”), substances that prevent thegrowth of HGPRT-deficient cells.

Preferred immortalized cell lines are those that fuse efficiently,support stable high-level production of antibody by the selectedantibody-producing cells, and are sensitive to a medium such as HATmedium. Among these myeloma cell lines are murine myeloma lines, such asthose derived from the MOPC-21 and MPC-11 mouse tumors available fromthe Salk Institute Cell Distribution Center, San Diego, Calif., andSP2/0 or X63-Ag8-653 cells available from the American Type CultureCollection (ATCC), Manassas, Va., USA. Human myeloma and mouse-humanheteromyeloma cell lines also have been described for the production ofhuman monoclonal antibodies (Kozbor, J Immunol 133:3001 (1984); Brodeur,et al., Monoclonal Antibody Production Techniques and Applications,Marcel Dekker, Inc, pp. 51-63 (1987)). The mouse myeloma cell line NSOmay also be used (European Collection of Cell Cultures, Salisbury,Wilshire, UK).

The culture medium in which hybridoma cells are grown is assayed forproduction of monoclonal antibodies directed against Notch3. The bindingspecificity of monoclonal antibodies produced by hybridoma cells may bedetermined by immunoprecipitation or by an in vitro binding assay, suchas radioimmunoassay (RIA) or enzyme-linked immunoabsorbent assay(ELISA). Such techniques are known in the art and within the skill ofthe artisan. The binding affinity of the monoclonal antibody to Notch3can, for example, be determined by a Scatchard analysis (MUNSON, et al.,Anal Biochem 107:220 (1980)).

After hybridoma cells are identified that produce antibodies of thedesired specificity, affinity, and/or activity, the clones may besubcloned by limiting dilution procedures and grown by standard methods(Goding, Monoclonal Antibodies: Principles and Practice, Academic Press,pp. 59-103 (1986)). Suitable culture media for this purpose include, forexample, Dulbecco's Modified Eagle's Medium (D-MEM) or RPMI-1640 medium.In addition, the hybridoma cells may be grown in vivo as ascites tumorsin an animal.

The monoclonal antibodies secreted by the subclones are suitablyseparated or isolated from the culture medium, ascites fluid, or serumby conventional immunoglobulin purification procedures such as, forexample, protein A-SEPHAROSE® affinity media, hydroxylaptitechromatography, gel exclusion chromatography, gel electrophoresis,dialysis, or affinity chromatography.

A variety of methods exist in the art for the production of monoclonalantibodies and thus, the invention is not limited to their soleproduction in hybridomas. For example, the monoclonal antibodies may bemade by recombinant DNA methods, such as those described in U.S. Pat.No. 4,816,567. In this context, the term “monoclonal antibody” refers toan antibody derived from a single eukaryotic, phage, or prokaryoticclone. DNA encoding the monoclonal antibodies of the invention isreadily isolated and sequenced using conventional procedures (e.g., byusing oligonucleotide probes that are capable of binding specifically togenes encoding the heavy and light chains of murine antibodies, or suchchains from human, humanized, or other sources) (Innis, et al. In PCRProtocols. A Guide to Methods and Applications, Academic (1990), Sanger,et al., Proc Natl Acad Sci 74:5463 (1977)). The hybridoma cells serve asa source of such DNA. Once isolated, the DNA may be placed intoexpression vectors, which are then transfected into host cells such asE. coli cells, NS0 cells, Simian COS cells, Chinese hamster ovary (CHO)cells, or myeloma cells that do not otherwise produce immunoglobulinprotein, to obtain the synthesis of monoclonal antibodies in therecombinant host cells. The DNA also may be modified, for example, bysubstituting the coding sequence for human heavy and light chainconstant domains in place of the homologous murine sequences (U.S. Pat.No. 4,816,567; Morrison, et al., Proc Natl Acad Sci USA 81:6851 (1984))or by covalently joining to the immunoglobulin coding sequence all orpart of the coding sequence for a non-immunoglobulin polypeptide. Such anon-immunoglobulin polypeptide can be substituted for the constantdomains of an antibody of the invention, or can be substituted for thevariable domains of one antigen-combining site of an antibody of theinvention to create a chimeric bivalent antibody.

The antibodies may be monovalent antibodies. Methods for preparingmonovalent antibodies are well known in the art. For example, one methodinvolves recombinant expression of immunoglobulin light chain andmodified heavy chain. The heavy chain is truncated generally at anypoint in the Fc region so as to prevent heavy chain cross-linking.Alternatively, the relevant cysteine residues are substituted withanother amino acid residue or are deleted so as to preventcross-linking.

Antibody fragments which recognize specific epitopes may be generated byknown techniques. Traditionally, these fragments were derived viaproteolytic digestion of intact antibodies (see, e.g., Morimoto, et al.,J Biochem Biophys Methods 24:107 (1992); Brennan, et al., Science 229:81(1985)). For example, Fab and F(ab′)₂ fragments of the invention may beproduced by proteolytic cleavage of immunoglobulin molecules, usingenzymes such as papain (to produce Fab fragments) or pepsin (to produceF(ab′)₂ fragments). F(ab′)₂ fragments contain the variable region, thelight chain constant region and the CH1 domain of the heavy chain.However, these fragments can now be produced directly by recombinanthost ells. For example, the antibody fragments can be isolated from anantibody phage library. Alternatively, F(ab′)₂-SH fragments can bedirectly recovered from E. coli and chemically coupled to form F(ab′)₂fragments (Carter, et al., Bio/Technology 10:163 (1992). According toanother approach, F(ab′)₂ fragments can be isolated directly fromrecombinant host cell culture. Other techniques for the production ofantibody fragments will be apparent to the skilled practitioner. Inother embodiments, the antibody of choice is a single chain Fv fragment(Fv) (PCT patent application WO 93/16185).

For some uses, including in vivo use of antibodies in humans and invitro detection assays, it may be preferable to use chimeric, humanized,or human antibodies. A chimeric antibody is a molecule in whichdifferent portions of the antibody are derived from different animalspecies, such as antibodies having a variable region derived from amurine monoclonal antibody and a human immunoglobulin constant region.Methods for producing chimeric antibodies are known in the art. Seee.g., Morrison, Science 229:1202 (1985); Oi, et al., BioTechniques 4:214(1986); Gillies, et al., J Immunol Methods 125:191 (1989); U.S. Pat.Nos. 5,807,715; 4,816,567; and 4,816397, which are incorporated hereinby reference in their entirety.

A humanized antibody is designed to have greater homology to a humanimmunoglobulin than animal-derived monoclonal antibodies. Humanizationis a technique for making a chimeric antibody wherein substantially lessthan an intact human variable domain has been substituted by thecorresponding sequence from a non-human species. Humanized antibodiesare antibody molecules generated in a non-human species that bind thedesired antigen having one or more complementarity determining regions(CDRs) from the non-human species and framework (FR) regions from ahuman immunoglobulin molecule. Often, framework residues in the humanframework regions will be substituted with the corresponding residuefrom the CDR donor antibody to alter, preferably improve, antigenbinding. These framework substitutions are identified by methods wellknown in the art, e.g., by modeling of the interactions of the CDR andframework residues to identify framework residues important for antigenbinding and sequence comparison to identify unusual framework residuesat particular positions. See, e.g., U.S. Pat. No. 5,585,089; Riechmann,et al., Nature 332:323 (1988), which are incorporated herein byreference in their entireties. Antibodies can be humanized using avariety of techniques known in the art including, for example,CDR-grafting (EP 239,400; PCT publication WO 91/09967; U.S. Pat. Nos.5,225,539; 5,530,101; and 5,585,089), veneering or resurfacing (EP592,106; EP 519,596; Padlan, Molecular Immunology 28:489 (1991);Studnicka, et al., Protein Engineering 7:805 (1994); Roguska, et al.,Proc Natl Acad Sci USA 91:969 (1994)), and chain shuffling (U.S. Pat.No. 5,565,332).

Generally, a humanized antibody has one or more amino acid residuesintroduced into it from a source that is non-human. These non-humanamino acid residues are often referred to as “import” residues, whichare typically taken from an “import” variable domain. Humanization canbe essentially performed following the methods of Winter and co-workers(Jones, et al., Nature 321:522 (1986); Riechmann, et al., Nature 332:323(1988); Verhoeyen, et al., Science 239:1534 (1988)), by substitutingnon-human CDRs or CDR sequences for the corresponding sequences of ahuman antibody. Accordingly, such “humanized” antibodies are chimericantibodies (U.S. Pat. No. 4,816,567), wherein substantially less than anintact human variable domain has been substituted by the correspondingsequence from a non-human species. In practice, humanized antibodies aretypically human antibodies in which some CDR residues and possible someFR residues are substituted from analogous sites in rodent antibodies.

It is further important that humanized antibodies retain high affinityfor the antigen and other favorable biological properties. To achievethis goal, according to a preferred method, humanized antibodies areprepared by a process of analysis of the parental sequences and variousconceptual humanized products using three-dimensional models of theparental and humanized sequences. Three-dimensional immunoglobulinmodels are commonly available and are familiar to those skilled in theart. Computer programs are available which illustrate and displayprobable three-dimensional conformational structures of selectedcandidate immunoglobulin sequences. Inspection of these displays permitsanalysis of the likely role of certain residues in the functioning ofthe candidate immunoglobulin sequence, i.e., the analysis of residuesthat influence the ability of the candidate immunoglobulin sequences,i.e., the analysis of residues that influence the ability of thecandidate immunoglobulin to bind its antigen. In this way, FR residuescan be selected and combined from the recipient and import sequences sothat the desired antibody characteristic, such as increased affinity forthe target antigen(s), is maximized, although it is the CDR residuesthat directly and most substantially influence antigen binding.

The choice of human variable domains, both light and heavy, to be usedin making the humanized antibodies is important to reduce antigenicity.According to an exemplary method, the so-called “best-fit” method, thesequence of the variable domain of a non-human antibody is screenedagainst the entire library of known human variable-domain sequences. Thehuman sequence which is closest to that of that of the non-human parentantibody is then accepted as the human FR for the humanized antibody(Sims, et al., J Immunol 151:2296 (1993); Chothia, et al., J Mol Biol196:901 (1987)). Another method uses a particular framework derived fromthe consensus sequence of all human antibodies of a particular subgroupof light or heavy chains. The same framework may be used for severaldifferent humanized antibodies (Carter, et al., Proc Natl Acad Sci USA89:4285 (1992); Presta, et al., J Immunol 151:2623 (1993)).

Completely human antibodies are particularly desirable for therapeutictreatment of human patients. Human antibodies can be made by a varietyof methods known in the art including phage display methods describedabove using antibody libraries derived from human immunoglobulinsequences. See also, U.S. Pat. Nos. 4,444,887 and 4,716,111; and PCTpublications WO 98/46645, WO 98/50433, WO 98/24893, WO 98/16654, WO96/34096, WO 96/33735, and WO 91/10741; each of which is incorporatedherein by reference in its entirety. The techniques of Cole, et al. andBoerder, et al. are also available for the preparation of humanmonoclonal antibodies (Cole, et al., Monoclonal Antibodies and CancerTherapy, Alan R. Riss (1985); and Boerner, et al., J Immunol 147:86(1991)).

Human antibodies can also be produced using transgenic mice which areincapable of expressing functional endogenous immunoglobulins, but whichcan express human immunoglobulin genes. For example, the human heavy andlight chain immunoglobulin gene complexes may be introduced randomly orby homologous recombination into mouse embryonic stem cells.Alternatively, the human variable region, constant region, and diversityregion may be introduced into mouse embryonic stem cells in addition tothe human heavy and light chain genes. The mouse heavy and light chainimmunoglobulin genes may be rendered non-functional separately orsimultaneously with the introduction of human immunoglobulin loci byhomologous recombination. In particular, homozygous deletion of the JHregion prevents endogenous antibody production. The modified embryonicstem cells are expanded and microinjected into blastocysts to producechimeric mice. The chimeric mice are then bred to produce homozygousoffspring which express human antibodies. See, e.g., Jakobovits, et al.,Proc Natl Acad Sci USA 90:2551 (1993); Jakobovits, et al., Nature362:255 (1993); Bruggermann, et al., Year in Immunol 7:33 (1993);Duchosal, et al., Nature 355:258 (1992)). The transgenic mice areimmunized in the normal fashion with a selected antigen, e.g., all or aportion of a polypeptide of the invention. Monoclonal antibodiesdirected against the antigen can be obtained from the immunized,transgenic mice using conventional hybridoma technology. The humanimmunoglobulin transgenes harbored by the transgenic mice rearrangeduring B cell differentiation, and subsequently undergo class switchingand somatic mutation. Thus, using such a technique, it is possible toproduce therapeutically useful IgG, IgA, IgM and IgE antibodies. For anoverview of this technology for producing human antibodies, see Lonberg,et al., Int Rev Immunol 13:65-93 (1995). For a detailed discussion ofthis technology for producing human antibodies and human monoclonalantibodies and protocols for producing such antibodies, see, e.g., PCTpublications WO 98/24893; WO 92/01047; WO 96/34096; WO 96/33735;European Patent No. 0 598 877; U.S. Pat. Nos. 5,413,923; 5,625,126;5,633,425; 5,569,825; 5,661,016; 5,545,806; 5,814,318; 5,885,793;5,916,771; and 5,939,598, which are incorporated by reference herein intheir entirety. In addition, companies such as Abgenix, Inc. (Freemont,Calif.), Genpharm (San Jose, Calif.), and Medarex, Inc. (Princeton,N.J.) can be engaged to provide human antibodies directed against aselected antigen using technology similar to that described above.

Also human mAbs could be made by immunizing mice transplanted with humanperipheral blood leukocytes, splenocytes or bone marrows (e.g., Triomatechniques of XTL). Completely human antibodies which recognize aselected epitope can be generated using a technique referred to as“guided selection.” In this approach a selected non-human monoclonalantibody, e.g., a mouse antibody, is used to guide the selection of acompletely human antibody recognizing the same epitope (Jespers, et al.,Bio/technology 12:899 (1988)).

Further, antibodies to the polypeptides of the invention can, in turn,be utilized to generate anti-idiotype antibodies that “mimic”polypeptides of the invention using techniques well known to thoseskilled in the art (See, e.g., Greenspan, et al., FASEB J 7:437 (1989);Nissinoff, J Immunol 147:2429 (1991)). For example, antibodies whichbind to and competitively inhibit polypeptide multimerization and/orbinding of a polypeptide of the invention to a ligand can be used togenerate anti-idiotypes that “mimic” the polypeptide multimerizationand/or binding domain and, as a consequence, bind to and neutralizepolypeptide and/or its ligand. Such neutralizing anti-idiotypes or Fabfragments of such anti-idiotypes can be used in therapeutic regimens toneutralize polypeptide ligand. For example, such anti-idiotypicantibodies can be used to bind a polypeptide of the invention and/or tobind its ligands/receptors, and thereby block its biological activity.

The antibodies of the present invention may be bispecific antibodies.Bispecific antibodies are monoclonal, preferably human or humanized,antibodies that have binding specificities for at least two differentantigens. In the present invention, one of the binding specificities maybe directed towards Notch3, the other may be for any other antigen, andpreferably for a cell-surface protein, receptor, receptor subunit,tissue-specific antigen, virally derived protein, virally encodedenvelope protein, bacterially derived protein, or bacterial surfaceprotein, etc.

Methods for making bispecific antibodies are well known. Traditionally,the recombinant production of bispecific antibodies is based on theco-expression of two immunoglobulin heavy-chain/light-chain pairs, wherethe two heavy chains have different specificities (Milstein, et al.,Nature 305:537 (1983)). Because of the random assortment ofimmunoglobulin heavy and light chains, these hybridomas (quadromas)produce a potential mixture of ten different antibody molecules, ofwhich only one has the correct bispecific structure. The purification ofthe correct molecule is usually accomplished by affinity chromatographysteps. Similar procedures are disclosed in WO 93/08829 and inTraunecker, et al., EMBO J 10:3655 (1991).

Antibody variable domains with the desired binding specificities(antibody-antigen combining sites) can be fused to immunoglobulinconstant domain sequences. The fusion preferably is with animmunoglobulin heavy-chain constant domain, comprising at least part ofthe hinge, CH2, and CH3 regions. It may have the first heavy-chainconstant region (CH1) containing the site necessary for light-chainbinding present in at least one of the fusions. DNAs encoding theimmunoglobulin heavy-chain fusions and, if desired, the immunoglobulinlight chain, are inserted into separate expression vectors, and areco-transformed into a suitable host organism. For further details ofgenerating bispecific antibodies see, for example Suresh, et al., MethIn Enzym 121:210 (1986).

Heteroconjugate antibodies are also contemplated by the presentinvention. Heteroconjugate antibodies are composed of two covalentlyjoined antibodies. Such antibodies have, for example, been proposed totarget immune system cells to unwanted cells (U.S. Pat. No. 4,676,980).It is contemplated that the antibodies may be prepared in vitro usingknown methods in synthetic protein chemistry, including those involvingcross-linking agents. For example, immunotoxins may be constructed usinga disulfide exchange reaction or by forming a thioester bond. Examplesof suitable reagents for this purpose include iminothiolate andmethyl-4-mercaptobutyrimidate and those disclosed, for example, in U.S.Pat. No. 4,676,980.

In addition, one can generate single-domain antibodies to Notch3.Examples of this technology have been described in WO94/25591 forantibodies derived from Camelidae heavy chain Ig, as well inUS2003/0130496 describing the isolation of single domain fully humanantibodies from phage libraries.

One can also create a single peptide chain binding molecules in whichthe heavy and light chain Fv regions are connected. Single chainantibodies (“scFv”) and the method of their construction are describedin U.S. Pat. No. 4,946,778. Alternatively, Fab can be constructed andexpressed by similar means. All of the wholly and partially humanantibodies are less immunogenic than wholly murine mAbs, and thefragments and single chain antibodies are also less immunogenic.

Antibodies or antibody fragments can be isolated from antibody phagelibraries generated using the techniques described in McCafferty, etal., Nature 348:552 (1990). Clarkson, et al., Nature 352:624 (1991) andMarks, et al., J Mol Biol 222:581 (1991) describe the isolation ofmurine and human antibodies, respectively, using phage libraries.Subsequent publications describe the production of high affinity (nMrange) human antibodies by chain shuffling (Marks, et al.,Bio/Technology 10:779 (1992)), as well as combinatorial infection and invivo recombination as a strategy for constructing very large phagelibraries (Waterhouse, et al., Nuc Acids Res 21:2265 (1993)). Thus,these techniques are viable alternatives to traditional monoclonalantibody hybridoma techniques for isolation of monoclonal antibodies.

The DNA also may be modified, for example, by substituting the codingsequence for human heavy- and light-chain constant domains in place ofthe homologous murine sequences (U.S. Pat. No. 4,816,567; Morrison, etal., Proc Natl Acad Sci USA 81:6851 (1984)).

Another alternative is to use electrical fusion rather than chemicalfusion to form hybridomas. This technique is well established. Insteadof fusion, one can also transform a B cell to make it immortal using,for example, an Epstein Barr Virus, or a transforming gene. See, e.g.,“Continuously Proliferating Human Cell Lines Synthesizing Antibody ofPredetermined Specificity,” Zurawaki, et al., in Monoclonal Antibodies,ed. by Kennett, et al., Plenum Press, pp. 19-33. (1980)). Anti-Notch3mAbs can be raised by immunizing rodents (e.g., mice, rats, hamsters,and guinea pigs) with Notch3 protein, fusion protein, or its fragmentsexpressed by either eukaryotic or prokaryotic systems. Other animals canbe used for immunization, e.g., non-human primates, transgenic miceexpressing immunoglobulins, and severe combined immunodeficient (SCID)mice transplanted with human B lymphocytes. Hybridomas can be generatedby conventional procedures by fusing B lymphocytes from the immunizedanimals with myeloma cells (e.g., Sp2/0 and NSO), as described earlier(Köhler, et al., Nature 256:495 (1975)). In addition, anti-Notch3antibodies can be generated by screening of recombinant single-chain Fvor Fab libraries from human B lymphocytes in phage-display systems. Thespecificity of the mAbs to Notch3 can be tested by ELISA, Westernimmunoblotting, or other immunochemical techniques. The inhibitoryactivity of the antibodies on complement activation can be assessed byhemolytic assays, using sensitized chicken or sheep RBCs for theclassical complement pathway. The hybridomas in the positive wells arecloned by limiting dilution. The antibodies are purified forcharacterization for specificity to human Notch3 by the assays describedabove.

Identification of Anti-Notch-3 Antibodies

The present invention provides antagonist monoclonal antibodies thatinhibit and neutralize the action of Notch3. In particular, theantibodies of the present invention bind to and inhibit the activationof Notch3. The antibodies of the present invention include theantibodies designated 256A-4 and 256A-8, which are disclosed herein. Thepresent invention also includes antibodies that bind to the same epitopeas one of these antibodies.

Candidate anti-Notch3 antibodies were tested by enzyme linkedimmunosorbent assay (ELISA), Western immunoblotting, or otherimmunochemical techniques. Assays performed to characterize theindividual antibodies are described in the Examples.

Antibodies of the invention include, but are not limited to, polyclonal,monoclonal, monovalent, bispecific, heteroconjugate, multispecific,human, humanized or chimeric antibodies, single chain antibodies,single-domain antibodies, Fab fragments, F(ab′) fragments, fragmentsproduced by a Fab expression library, anti-idiotypic (anti-Id)antibodies (including, e.g., anti-Id antibodies to antibodies of theinvention), and epitope-binding fragments of any of the above.

The antibodies may be human antigen-binding antibody fragments of thepresent invention and include, but are not limited to, Fab, Fab′ andF(ab′)₂, Fd, single-chain Fvs (scFv), single-chain antibodies,disulfide-linked Fvs (sdFv) and single-domain antibodies comprisingeither a VL or VH domain. Antigen-binding antibody fragments, includingsingle-chain antibodies, may comprise the variable region(s) alone or incombination with the entirety or a portion of the following: hingeregion, CH1, CH2, and CH3 domains. Also included in the invention areantigen-binding fragments comprising any combination of variableregion(s) with a hinge region, CH1, CH2, and CH3 domains. The antibodiesof the invention may be from any animal origin including birds andmammals. Preferably, the antibodies are from human, non-human primates,rodents (e.g., mouse and rat), donkey, sheep, rabbit, goat, guinea pig,camel, horse, or chicken.

As used herein, “human” antibodies” include antibodies having the aminoacid sequence of a human immunoglobulin and include antibodies isolatedfrom human immunoglobulin libraries or from animals transgenic for oneor more human immunoglobulin and that do not express endogenousimmunoglobulins, as described infra and, for example in, U.S. Pat. No.5,939,598 by Kucherlapati, et al.

The antibodies of the present invention may be monospecific, bispecific,trispecific or of greater multispecificity. Multispecific antibodies maybe specific for different epitopes of Notch3 or may be specific for bothNotch3 as well as for a heterologous epitope, such as a heterologouspolypeptide or solid support material. See, e.g., PCT publications WO93/17715; WO 92/08802; WO 91/00360; WO 92/05793; Tutt, et al., J Immunol147:60 (1991); U.S. Pat. Nos. 4,474,893; 4,714,681; 4,925,648;5,573,920; 5,601,819; Kostelny, et al., J Immunol 148:1547 (1992).

Antibodies of the present invention may be described or specified interms of the epitope(s) or portion(s) of Notch3 which they recognize orspecifically bind. The epitope(s) or polypeptide portion(s) may bespecified as described herein, e.g., by N-terminal and C-terminalpositions, by size in contiguous amino acid residues, or listed in theTables and Figures.

Antibodies of the present invention may also be described or specifiedin terms of their cross-reactivity. Antibodies that bind Notch3polypeptides, which have at least 95%, at least 90%, at least 85%, atleast 80%, at least 75%, at least 70%, at least 65%, at least 60%, atleast 55%, and at least 50% identity (as calculated using methods knownin the art and described herein) to Notch3 are also included in thepresent invention. Anti-Notch3 antibodies may also bind with a K_(D) ofless than about 10⁻⁷ M, less than about 10⁻⁶ M, or less than about 10⁻⁵M to other proteins, such as anti-Notch3 antibodies from species otherthan that against which the anti-Notch3 antibody is directed.

In specific embodiments, antibodies of the present invention cross-reactwith monkey homologues of human Notch3 and the corresponding epitopesthereof. In a specific embodiment, the above-described cross-reactivityis with respect to any single specific antigenic or immunogenicpolypeptide, or combination(s) of the specific antigenic and/orimmunogenic polypeptides disclosed herein.

Further included in the present invention are antibodies which bindpolypeptides encoded by polynucleotides which hybridize to apolynucleotide encoding Notch3 under stringent hybridization conditions.Antibodies of the present invention may also be described or specifiedin terms of their binding affinity to a polypeptide of the invention.Preferred binding affinities include those with an equilibriumdissociation constant or K_(D) from 10⁻⁸ to 10⁻¹⁵ M, 10⁻⁸ to 10⁻¹² M,10⁻⁸ to 10⁻¹⁰ M, or 10⁻¹⁰ to 10⁻¹² M. The invention also providesantibodies that competitively inhibit binding of an antibody to anepitope of the invention as determined by any method known in the artfor determining competitive binding, for example, the immunoassaysdescribed herein. In preferred embodiments, the antibody competitivelyinhibits binding to the epitope by at least 95%, at least 90%, at least85%, at least 80%, at least 75%, at least 70%, at least 60%, or at least50%.

Vectors and Host Cells

In another aspect, the present invention provides isolated nucleic acidsequences encoding an antibody as disclosed herein, vector constructscomprising a nucleotide sequence encoding the antibodies of the presentinvention, host cells comprising such a vector, and recombinanttechniques for the production of the antibody.

For recombinant production of an antibody, the nucleic acid encoding itis isolated and inserted into a replicable vector for further cloning(amplification of the DNA) or for expression. DNA encoding the antibodyis readily isolated and sequenced using conventional procedures (e.g.,by using oligonucleotide probes that are capable of binding specificallyto genes encoding the heavy and light chains of the antibody). Standardtechniques for cloning and transformation may be used in the preparationof cell lines expressing the antibodies of the present invention.

Vectors

Many vectors are available. The vector components generally include, butare not limited to, one or more of the following: a signal sequence, anorigin of replication, one or more marker genes, an enhancer element, apromoter, and a transcription termination sequence. Recombinantexpression vectors containing a nucleotide sequence encoding theantibodies of the present invention can be prepared using well knowntechniques. The expression vectors include a nucleotide sequenceoperably linked to suitable transcriptional or translational regulatorynucleotide sequences such as those derived from mammalian, microbial,viral, or insect genes. Examples of regulatory sequences includetranscriptional promoters, operators, enhancers, mRNA ribosomal bindingsites, and/or other appropriate sequences which control transcriptionand translation initiation and termination. Nucleotide sequences are“operably linked” when the regulatory sequence functionally relates tothe nucleotide sequence for the appropriate polypeptide. Thus, apromoter nucleotide sequence is operably linked to, e.g., the antibodyheavy chain sequence if the promoter nucleotide sequence controls thetranscription of the appropriate nucleotide sequence.

In addition, sequences encoding appropriate signal peptides that are notnaturally associated with antibody heavy and/or light chain sequencescan be incorporated into expression vectors. For example, a nucleotidesequence for a signal peptide (secretory leader) may be fused in-frameto the polypeptide sequence so that the antibody is secreted to theperiplasmic space or into the medium. A signal peptide that isfunctional in the intended host cells enhances extracellular secretionof the appropriate antibody. The signal peptide may be cleaved from thepolypeptide upon secretion of antibody from the cell. Examples of suchsecretory signals are well known and include, e.g., those described inU.S. Pat. Nos. 5,698,435; 5,698,417; and 6,204,023.

The vector may be a plasmid vector, a single or double-stranded phagevector, or a single or double-stranded RNA or DNA viral vector. Suchvectors may be introduced into cells as polynucleotides by well knowntechniques for introducing DNA and RNA into cells. The vectors, in thecase of phage and viral vectors also may be introduced into cells aspackaged or encapsulated virus by well known techniques for infectionand transduction. Viral vectors may be replication competent orreplication defective. In the latter case, viral propagation generallywill occur only in complementing host cells. Cell-free translationsystems may also be employed to produce the protein using RNAs derivedfrom the present DNA constructs. Such vectors may include the nucleotidesequence encoding the constant region of the antibody molecule (see,e.g., PCT Publications WO 86/05807 and WO 89/01036; and U.S. Pat. No.5,122,464) and the variable domain of the antibody may be cloned intosuch a vector for expression of the entire heavy or light chain.

Host Cells

The antibodies of the present invention can be expressed from anysuitable host cell. Examples of host cells useful in the presentinvention include prokaryotic, yeast, or higher eukaryotic cells andinclude but are not limited to microorganisms such as bacteria (e.g., E.coli, B. subtilis) transformed with recombinant bacteriophage DNA,plasmid DNA or cosmid DNA expression vectors containing antibody codingsequences; yeast (e.g., Saccharomyces, Pichia) transformed withrecombinant yeast expression vectors containing antibody codingsequences; insect cell systems infected with recombinant virusexpression vectors (e.g., Baculovirus) containing antibody codingsequences; plant cell systems infected with recombinant virus expressionvectors (e.g., cauliflower mosaic virus, CaMV; tobacco mosaic virus,TMV) or transformed with recombinant plasmid expression vectors (e.g.,Ti plasmid) containing antibody coding sequences; or mammalian cellsystems (e.g., COS, CHO, BHK, 293, 3T3 cells) harboring recombinantexpression constructs containing promoters derived from the genome ofmammalian cells (e.g., metallothionein promoter) or from mammalianviruses (e.g., the adenovirus late promoter; the vaccinia virus 7.5Kpromoter).

Prokaryotes useful as host cells in the present invention include gramnegative or gram positive organisms such as E. coli, B. subtilis,Enterobacter, Erwinia, Klebsiella, Proteus, Salmonella, Serratia, andShigella, as well as Bacilli, Pseudomonas, and Streptomyces. Onepreferred E. coli cloning host is E. coli 294 (ATCC 31,446), althoughother strains such as E. coli B, E. coli X1776 (ATCC 31,537), and E.coli W3110 (ATCC 27,325) are suitable. These examples are illustrativerather than limiting.

Expression vectors for use in prokaryotic host cells generally compriseone or more phenotypic selectable marker genes. A phenotypic selectablemarker gene is, for example, a gene encoding a protein that confersantibiotic resistance or that supplies an autotrophic requirement.Examples of useful expression vectors for prokaryotic host cells includethose derived from commercially available plasmids such as the pKK223-3vector (Pharmacia Fine Chemicals, Uppsala, Sweden), PGEM® 1 vector(Promega Biotec, Madison, Wis., USA), and the pET (Novagen, Madison,Wis., USA) and pRSET (Invitrogen, Carlsbad, Calif.) series of vectors(Studier, J Mol Biol 219:37 (1991); Schoepfer, Gene 124:83 (1993)).Promoter sequences commonly used for recombinant prokaryotic host cellexpression vectors include T7, (Rosenberg, et al., Gene 56:125 (1987)),β-lactamase (penicillinase), lactose promoter system (Chang, et al.,Nature 275:615 (1978); Goeddel, et al., Nature 281:544 (1979)),tryptophan (trp) promoter system (Goeddel, et al., Nucl Acids Res 8:4057(1980)), and tac promoter (Sambrook, et al., Molecular Cloning, ALaboratory Manual, 2nd ed., Cold Spring Harbor Laboratory (1990)).

Yeasts or filamentous fungi useful in the present invention includethose from the genus Saccharomyces, Pichia, Actinomycetes,Kluyveromyces, Schizosaccharomyces, Candida, Trichoderma, Neurospora,and filamentous fungi such as Neurospora, Penicillium, Tolypocladium,and Aspergillus. Yeast vectors will often contain an origin ofreplication sequence from a 2μ yeast plasmid, an autonomouslyreplicating sequence (ARS), a promoter region, sequences forpolyadenylation, sequences for transcription termination, and aselectable marker gene. Suitable promoter sequences for yeast vectorsinclude, among others, promoters for metallothionein, 3-phosphoglyceratekinase (Hitzeman, et al., J Biol Chem 255:2073 (1980)) or otherglycolytic enzymes (Holland, et al., Biochem 17:4900 (1978)) such asenolase, glyceraldehyde-3-phosphate dehydrogenase, hexokinase, pyruvatedecarboxylase, phosphofructokinase, glucose-6-phosphate isomerase,3-phosphoglycerate mutase, pyruvate kinase, triosephosphate isomerase,phosphoglucose isomerase, and glucokinase. Other suitable vectors andpromoters for use in yeast expression are further described in Fleer, etal., Gene 107:285 (1991). Other suitable promoters and vectors for yeastand yeast transformation protocols are well known in the art. Yeasttransformation protocols are well known. One such protocol is describedby Hinnen, et al., Proc Natl Acad Sci 75:1929 (1978). The Hinnenprotocol selects for Trp⁺ transformants in a selective medium.

Mammalian or insect host cell culture systems may also be employed toexpress recombinant antibodies. In principle, any higher eukaryotic cellculture is workable, whether from vertebrate or invertebrate culture.Examples of invertebrate cells include plant and insect cells (Luckow,et al., Bio/Technology 6:47 (1988); Miller, et al., GeneticsEngineering, Setlow, et al., eds. Vol. 8, pp. 277-9, Plenam Publishing(1986); Mseda, et al., Nature 315:592 (1985)). For example, Baculovirussystems may be used for production of heterologous proteins. In aninsect system, Autographa californica nuclear polyhedrosis virus (AcNPV)may be used as a vector to express foreign genes. The virus grows inSpodoptera frugiperda cells. The antibody coding sequence may be clonedindividually into non-essential regions (for example the polyhedringene) of the virus and placed under control of an AcNPV promoter (forexample the polyhedrin promoter). Other hosts that have been identifiedinclude Aedes, Drosophila melanogaster, and Bombyx mori. A variety ofviral strains for transfection are publicly available, e.g., the L-1variant of AcNPV and the Bm-5 strain of Bombyx mori NPV, and suchviruses may be used as the virus herein according to the presentinvention, particularly for transfection of Spodoptera frugiperda cells.Moreover, plant cells cultures of cotton, corn, potato, soybean,petunia, tomato, and tobacco and also be utilized as hosts.

Vertebrate cells, and propagation of vertebrate cells, in culture(tissue culture) has become a routine procedure. See Tissue Culture,Kruse, et al., eds., Academic Press (1973). Examples of useful mammalianhost cell lines are monkey kidney; human embryonic kidney line; babyhamster kidney cells; Chinese hamster ovary cells/−DHFR (CHO, Urlaub, etal., Proc Natl Acad Sci USA 77:4216 (1980)); mouse sertoli cells; humancervical carcinoma cells (HELA); canine kidney cells; human lung cells;human liver cells; mouse mammary tumor; and NS0 cells.

Host cells are transformed with the above-described vectors for antibodyproduction and cultured in conventional nutrient media modified asappropriate for inducing promoters, transcriptional and translationalcontrol sequences, selecting transformants, or amplifying the genesencoding the desired sequences. Commonly used promoter sequences andenhancer sequences are derived from polyoma virus, Adenovirus 2, Simianvirus 40 (SV40), and human cytomegalovirus (CMV). DNA sequences derivedfrom the SV40 viral genome may be used to provide other genetic elementsfor expression of a structural gene sequence in a mammalian host cell,e.g., SV40 origin, early and late promoter, enhancer, splice, andpolyadenylation sites. Viral early and late promoters are particularlyuseful because both are easily obtained from a viral genome as afragment which may also contain a viral origin of replication. Exemplaryexpression vectors for use in mammalian host cells are commerciallyavailable.

The host cells used to produce an antibody of this invention may becultured in a variety of media. Commercially available media such asHam's F10 (Sigma, St Louis, Mo.), Minimal Essential Medium (MEM, Sigma,St Louis, Mo.), RPMI-1640 (Sigma, St Louis, Mo.), and Dulbecco'sModified Eagle's Medium (DMEM, Sigma, St Louis, Mo.) are suitable forculturing host cells. In addition, any of the media described in Ham, etal., Meth Enzymol 58:44 (1979), Barnes, et al., Anal Biochem 102:255(1980), and U.S. Pat. No. 4,767,704; 4,657,866; 4,560,655; 5,122,469;5,712,163; or 6,048,728 may be used as culture media for the host cells.Any of these media may be supplemented as necessary with hormones and/orother growth factors (such as insulin, transferrin, or epidermal growthfactor), salts (such as X-chlorides, where X is sodium, calcium,magnesium; and phosphates), buffers (such as HEPES), nucleotides (suchas adenosine and thymidine), antibiotics (such as gentamicin), traceelements (defined as inorganic compounds usually present at finalconcentrations in the micromolar range), and glucose or an equivalentenergy source. Any other necessary supplements may also be included atappropriate concentrations that would be known to those skilled in theart. The culture conditions, such as temperature, pH, and the like, arethose previously used with the host cell selected for expression, andwill be apparent to the ordinarily skilled artisan.

Polynucleotides Encoding Antibodies

The invention further provides polynucleotides or nucleic acids, e.g.,DNA, comprising a nucleotide sequence encoding an antibody of theinvention and fragments thereof. Exemplary polynucleotides include thoseencoding antibody chains comprising one or more of the amino acidsequences described herein. The invention also encompassespolynucleotides that hybridize under stringent or lower stringencyhybridization conditions to polynucleotides that encode an antibody ofthe present invention.

The polynucleotides may be obtained, and the nucleotide sequence of thepolynucleotides determined, by any method known in the art. For example,if the nucleotide sequence of the antibody is known, a polynucleotideencoding the antibody may be assembled from chemically synthesizedoligonucleotides (e.g., as described in Kutmeier, et al., Bio/Techniques17:242 (1994)), which, briefly, involves the synthesis of overlappingoligonucleotides containing portions of the sequence encoding theantibody, annealing and ligating of those oligonucleotides, and thenamplification of the ligated oligonucleotides by PCR.

Alternatively, a polynucleotide encoding an antibody may be generatedfrom nucleic acid from a suitable source. If a clone containing anucleic acid encoding a particular antibody is not available, but thesequence of the antibody molecule is known, a nucleic acid encoding theimmunoglobulin may be chemically synthesized or obtained from a suitablesource (e.g., an antibody cDNA library, or a cDNA library generatedfrom, or nucleic acid, preferably poly A⁺ RNA, isolated from, any tissueor cells expressing the antibody, such as hybridoma cells selected toexpress an antibody of the invention) by PCR amplification usingsynthetic primers hybridizable to the 3′ and 5′ ends of the sequence orby cloning using an oligonucleotide probe specific for the particulargene sequence to identify, e.g., a cDNA clone from a cDNA library thatencodes the antibody. Amplified nucleic acids generated by PCR may thenbe cloned into replicable cloning vectors using any method well known inthe art.

Once the nucleotide sequence and corresponding amino acid sequence ofthe antibody is determined, the nucleotide sequence of the antibody maybe manipulated using methods well known in the art for the manipulationof nucleotide sequences, e.g., recombinant DNA techniques, site directedmutagenesis, PCR, etc. (see, for example, the techniques described inSambrook, et al., Molecular Cloning, A Laboratory Manual, 2nd ed., ColdSpring Harbor Laboratory (1990); Ausubel, et al., eds., CurrentProtocols in Molecular Biology, John Wiley & Sons (1998), which are bothincorporated by reference herein in their entireties), to generateantibodies having a different amino acid sequence, for example to createamino acid substitutions, deletions, and/or insertions.

In a specific embodiment, the amino acid sequence of the heavy and/orlight chain variable domains may be inspected to identify the sequencesof the CDRs by well known methods, e.g., by comparison to known aminoacid sequences of other heavy and light chain variable regions todetermine the regions of sequence hypervariability. Using routinerecombinant DNA techniques, one or more of the CDRs may be insertedwithin framework regions, e.g., into human framework regions to humanizea non-human antibody, as described supra. The framework regions may benaturally occurring or consensus framework regions, and preferably humanframework regions (see, e.g., Chothia, et al., J Mol Biol 278: 457(1998) for a listing of human framework regions). Preferably, thepolynucleotide generated by the combination of the framework regions andCDRs encodes an antibody that specifically binds a polypeptide of theinvention. Preferably, as discussed supra, one or more amino acidsubstitutions may be made within the framework regions, and, preferably,the amino acid substitutions improve binding of the antibody to itsantigen. Additionally, such methods may be used to make amino acidsubstitutions or deletions of one or more variable region cysteineresidues participating in an intrachain disulfide bond to generateantibody molecules lacking one or more intrachain disulfide bonds. Otheralterations to the polynucleotide are encompassed by the presentinvention and within the skill of the art.

In addition, techniques developed for the production of “chimericantibodies” (Morrison, et al., Proc Natl Acad Sci 81:851 (1984);Neuberger, et al., Nature 312:604 (1984); Takeda, et al., Nature 314:452(1985)) by splicing genes from a mouse antibody molecule of appropriateantigen specificity together with genes from a human antibody moleculeof appropriate biological activity can be used. As described supra, achimeric antibody is a molecule in which different portions are derivedfrom different animal species, such as those having a variable regionderived from a murine mAb and a human immunoglobulin constant region,e.g., humanized antibodies.

Alternatively, techniques described for the production of single chainantibodies (U.S. Pat. No. 4,946,778; Bird, Science 242:423 (1988);Huston, et al., Proc Natl Acad Sci USA 85:5879 (1988); and Ward, et al.,Nature 334:544 (1989)) can be adapted to produce single chainantibodies. Single chain antibodies are formed by linking the heavy andlight chain fragments of the Fv region via an amino acid bridge,resulting in a single chain polypeptide. Techniques for the assembly offunctional Fv fragments in E. coli may also be used (Skerra, et al.,Science 242:1038 (1988)).

Methods of Producing Anti-Notch3 Antibodies

The antibodies of the invention can be produced by any method known inthe art for the synthesis of antibodies, in particular, by chemicalsynthesis or preferably, by recombinant expression techniques.

Recombinant expression of an antibody of the invention, or fragment,derivative, or analog thereof, (e.g., a heavy or light chain of anantibody of the invention or a single chain antibody of the invention),requires construction of an expression vector containing apolynucleotide that encodes the antibody or a fragment of the antibody.Once a polynucleotide encoding an antibody molecule has been obtained,the vector for the production of the antibody may be produced byrecombinant DNA technology. An expression vector is constructedcontaining antibody coding sequences and appropriate transcriptional andtranslational control signals. These methods include, for example, invitro recombinant DNA techniques, synthetic techniques, and in vivogenetic recombination.

The expression vector is transferred to a host cell by conventionaltechniques and the transfected cells are then cultured by conventionaltechniques to produce an antibody of the invention. In one aspect of theinvention, vectors encoding both the heavy and light chains may beco-expressed in the host cell for expression of the entireimmunoglobulin molecule, as detailed below.

A variety of host-expression vector systems may be utilized to expressthe antibody molecules of the invention as described above. Suchhost-expression systems represent vehicles by which the coding sequencesof interest may be produced and subsequently purified, but alsorepresent cells which may, when transformed or transfected with theappropriate nucleotide coding sequences, express an antibody molecule ofthe invention in situ. Bacterial cells such as E. coli, and eukaryoticcells are commonly used for the expression of a recombinant antibodymolecule, especially for the expression of whole recombinant antibodymolecule. For example, mammalian cells such as CHO, in conjunction witha vector such as the major intermediate early gene promoter element fromhuman cytomegalovirus, are an effective expression system for antibodies(Foecking, et al., Gene 45:101 (1986); Cockett, et al., Bio/Technology8:2 (1990)).

In addition, a host cell strain may be chosen which modulates theexpression of the inserted sequences, or modifies and processes the geneproduct in the specific fashion desired. Such modifications (e.g.,glycosylation) and processing (e.g., cleavage) of protein products maybe important for the function of the protein. Different host cells havecharacteristic and specific mechanisms for the post-translationalprocessing and modification of proteins and gene products. Appropriatecell lines or host systems can be chosen to ensure the correctmodification and processing of the foreign protein expressed. To thisend, eukaryotic host cells which possess the cellular machinery forproper processing of the primary transcript, glycosylation, andphosphorylation of the gene product may be used. Such mammalian hostcells include, but are not limited to, CHO, COS, 293, 3T3, or myelomacells.

For long-term, high-yield production of recombinant proteins, stableexpression is preferred. For example, cell lines which stably expressthe antibody molecule may be engineered. Rather than using expressionvectors which contain viral origins of replication, host cells can betransformed with DNA controlled by appropriate expression controlelements (e.g., promoter, enhancer, sequences, transcriptionterminators, polyadenylation sites, etc.), and a selectable marker.Following the introduction of the foreign DNA, engineered cells may beallowed to grow for one to two days in an enriched media, and then areswitched to a selective media. The selectable marker in the recombinantplasmid confers resistance to the selection and allows cells to stablyintegrate the plasmid into their chromosomes and grow to form foci whichin turn can be cloned and expanded into cell lines. This method mayadvantageously be used to engineer cell lines which express the antibodymolecule. Such engineered cell lines may be particularly useful inscreening and evaluation of compounds that interact directly orindirectly with the antibody molecule.

A number of selection systems may be used, including but not limited tothe herpes simplex virus thymidine kinase (Wigler, et al., Cell 11:223(1977)), hypoxanthine-guanine phosphoribosyltransferase (Szybalska, etal., Proc Natl Acad Sci USA 48:202 (1992)), and adeninephosphoribosyltransferase (Lowy, et al., Cell 22:817 (1980)) genes canbe employed in tk, hgprt or aprt-cells, respectively. Also,antimetabolite resistance can be used as the basis of selection for thefollowing genes: dhfr, which confers resistance to methotrexate (Wigler,et al., Proc Natl Acad Sci USA 77:357 (1980); O'Hare, et al., Proc NatlAcad Sci USA 78:1527 (1981)); gpt, which confers resistance tomycophenolic acid (Mulligan, et al., Proc Natl Acad Sci USA 78:2072(1981)); neo, which confers resistance to the aminoglycoside G-418 (Wu,et al., Biotherapy 3:87 (1991)); and hygro, which confers resistance tohygromycin (Santerre, et al., Gene 30:147 (1984)). Methods commonlyknown in the art of recombinant DNA technology may be routinely appliedto select the desired recombinant clone, and such methods are described,for example, in Ausubel, et al., eds., Current Protocols in MolecularBiology, John Wiley & Sons (1993); Kriegler, Gene Transfer andExpression, A Laboratory Manual, Stockton Press (1990); and in Chapters12 and 13, Dracopoli, et al., eds, Current Protocols in Human Genetics,John Wiley & Sons (1994); Colberre-Garapin, et al., J Mol Biol 150:1(1981), which are incorporated by reference herein in their entireties.

The expression levels of an antibody molecule can be increased by vectoramplification (for a review, see Bebbington, et al., “The use of vectorsbased on gene amplification for the expression of cloned genes inmammalian cells,” DNA Cloning, Vol. 3. Academic Press (1987)). When amarker in the vector system expressing antibody is amplifiable, increasein the level of inhibitor present in culture of host cell will increasethe number of copies of the marker gene. Since the amplified region isassociated with the antibody gene, production of the antibody will alsoincrease (Crouse, et al., Mol Cell Biol 3:257 (1983)).

The host cell may be co-transfected with two expression vectors of theinvention, the first vector encoding a heavy chain derived polypeptideand the second vector encoding a light chain derived polypeptide. Thetwo vectors may contain identical selectable markers which enable equalexpression of heavy and light chain polypeptides. Alternatively, asingle vector may be used which encodes, and is capable of expressing,both heavy and light chain polypeptides. In such situations, the lightchain should be placed before the heavy chain to avoid an excess oftoxic free heavy chain (Proudfoot, Nature 322:52 (1986); Kohler, ProcNatl Acad Sci USA 77:2197 (1980)). The coding sequences for the heavyand light chains may comprise cDNA or genomic DNA.

Once an antibody molecule of the invention has been produced by ananimal, chemically synthesized, or recombinantly expressed, it may bepurified by any method known in the art for purification of animmunoglobulin molecule, for example, by chromatography (e.g., ionexchange, affinity, particularly by affinity for the specific antigenafter Protein A, and size-exclusion chromatography), centrifugation,differential solubility, or by any other standard technique for thepurification of proteins. In addition, the antibodies of the presentinvention or fragments thereof can be fused to heterologous polypeptidesequences described herein or otherwise known in the art, to facilitatepurification.

The present invention encompasses antibodies recombinantly fused orchemically conjugated (including both covalently and non-covalentlyconjugations) to a polypeptide. Fused or conjugated antibodies of thepresent invention may be used for ease in purification. See e.g., PCTpublication WO 93/21232; EP 439,095; Naramura, et al., Immunol Lett39:91 (1994); U.S. Pat. No. 5,474,981; Gillies, et al., Proc Natl AcadSci USA 89:1428 (1992); Fell, et al., J Immunol 146:2446 (1991), whichare incorporated by reference in their entireties.

Moreover, the antibodies or fragments thereof of the present inventioncan be fused to marker sequences, such as a peptide to facilitatepurification. In preferred embodiments, the marker amino acid sequenceis a hexa-histidine peptide, such as the tag provided in a pQE vector(QIAGEN, Inc., Valencia, Calif.), among others, many of which arecommercially available. As described in Gentz, et al., Proc Natl AcadSci USA 86:821 (1989), for instance, hexa-histidine provides forconvenient purification of the fusion protein. Other peptide tags usefulfor purification include, but are not limited to, the “HA” tag, whichcorresponds to an epitope derived from the influenza hemagglutininprotein (Wilson, et al., Cell 37:767 (1984)) and the “flag” tag.

Antibody Purification

When using recombinant techniques, an antibody can be producedintracellularly, in the periplasmic space, or directly secreted into themedium. If the antibody is produced intracellularly, as a first step,the particulate debris, either host cells or lysed fragments, may beremoved, for example, by centrifugation or ultrafiltration. Carter, etal., Bio/Technology 10:163 (1992) describe a procedure for isolatingantibodies which are secreted to the periplasmic space of E. coli.Briefly, cell paste is thawed in the presence of sodium acetate (pH3.5), EDTA, and phenylmethylsulfonylfluoride (PMSF) over about 30minutes. Cell debris can be removed by centrifugation. Where theantibody is secreted into the medium, supernatants from such expressionsystems are generally first concentrated using a commercially availableprotein concentration filter, for example, an Amicon or MilliporePellicon ultrafiltration unit. A protease inhibitor such as PMSF may beincluded in any of the foregoing steps to inhibit proteolysis andantibiotics may be included to prevent the growth of adventitiouscontaminants.

The antibody composition prepared from the cells can be purified using,for example, hydroxylapatite chromatography, gel electrophoresis,dialysis, and affinity chromatography, with affinity chromatographybeing the preferred purification technique. The suitability of protein Aas an affinity ligand depends on the species and isotype of anyimmunoglobulin Fc domain that is present in the antibody. Protein A canbe used to purify antibodies that are based on human IgG1, IgG2 or IgG4heavy chains (Lindmark, et al., J Immunol Meth 62:1 (1983)). Protein Gis recommended for all mouse isotypes and for human IgG3 (Guss, et al.,EMBO J 5:1567 (1986)). The matrix to which the affinity ligand isattached is most often agarose, but other matrices are available.Mechanically stable matrices such as controlled pore glass orpoly(styrenedivinyl)benzene allow for faster flow rates and shorterprocessing times than can be achieved with agarose. Where the antibodycomprises a CH3 domain, the Bakerbond ABX™ resin (J. T. Baker;Phillipsburg, N.J.) is useful for purification. Other techniques forprotein purification such as fractionation on an ion-exchange column,ethanol precipitation, Reverse Phase HPLC, chromatography on silica,chromatography on heparin SEPHAROSE® chromatography on an anion orcation exchange resin (such as a polyaspartic acid column),chromatofocusing, SDS-PAGE, and ammonium sulfate precipitation are alsoavailable depending on the antibody to be recovered.

Following any preliminary purification step(s), the mixture comprisingthe antibody of interest and contaminants may be subjected to low pHhydrophobic interaction chromatography using an elution buffer at a pHbetween about 2.5-4.5, preferably performed at low salt concentrations(e.g., from about 0-0.25M salt).

Pharmaceutical Formulation

Therapeutic formulations of the polypeptide or antibody may be preparedfor storage as lyophilized formulations or aqueous solutions by mixingthe polypeptide having the desired degree of purity with optional“pharmaceutically-acceptable” carriers, excipients or stabilizerstypically employed in the art (all of which are termed “excipients”),i.e., buffering agents, stabilizing agents, preservatives, isotonifiers,non-ionic detergents, antioxidants, and other miscellaneous additives.See Remington's Pharmaceutical Sciences, 16th edition, Osol, Ed. (1980).Such additives must be nontoxic to the recipients at the dosages andconcentrations employed.

Buffering agents help to maintain the pH in the range which approximatesphysiological conditions. They are preferably present at concentrationranging from about 2 mM to about 50 mM. Suitable buffering agents foruse with the present invention include both organic and inorganic acidsand salts thereof such as citrate buffers (e.g., monosodiumcitrate-disodium citrate mixture, citric acid-trisodium citrate mixture,citric acid-monosodium citrate mixture, etc.), succinate buffers (e.g.,succinic acid-monosodium succinate mixture, succinic acid-sodiumhydroxide mixture, succinic acid-disodium succinate mixture, etc.),tartrate buffers (e.g., tartaric acid-sodium tartrate mixture, tartaricacid-potassium tartrate mixture, tartaric acid-sodium hydroxide mixture,etc.), fumarate buffers (e.g., fumaric acid-monosodium fumarate mixture,fumaric acid-disodium fumarate mixture, monosodium fumarate-disodiumfumarate mixture, etc.), gluconate buffers (e.g., gluconic acid-sodiumglyconate mixture, gluconic acid-sodium hydroxide mixture, gluconicacid-potassium glyconate mixture, etc.), oxalate buffer (e.g., oxalicacid-sodium oxalate mixture, oxalic acid-sodium hydroxide mixture,oxalic acid-potassium oxalate mixture, etc.), lactate buffers (e.g.,lactic acid-sodium lactate mixture, lactic acid-sodium hydroxidemixture, lactic acid-potassium lactate mixture, etc.) and acetatebuffers (e.g., acetic acid-sodium acetate mixture, acetic acid-sodiumhydroxide mixture, etc.). Additionally, there may be mentioned phosphatebuffers, histidine buffers and trimethylamine salts such as Tris.

Preservatives may be added to retard microbial growth, and may be addedin amounts ranging from 0.2%-1% (w/v). Suitable preservatives for usewith the present invention include phenol, benzyl alcohol, meta-cresol,methyl paraben, propyl paraben, octadecyldimethylbenzyl ammoniumchloride, benzalconium halides (e.g., chloride, bromide, iodide),hexamethonium chloride, and alkyl parabens such as methyl or propylparaben, catechol, resorcinol, cyclohexanol, and 3-pentanol.

Isotonicifiers sometimes known as “stabilizers” may be added to ensureisotonicity of liquid compositions of the present invention and includepolyhydric sugar alcohols, preferably trihydric or higher sugaralcohols, such as glycerin, erythritol, arabitol, xylitol, sorbitol andmannitol.

Stabilizers refer to a broad category of excipients which can range infunction from a bulking agent to an additive which solubilizes thetherapeutic agent or helps to prevent denaturation or adherence to thecontainer wall. Typical stabilizers can be polyhydric sugar alcohols(enumerated above); amino acids such as arginine, lysine, glycine,glutamine, asparagine, histidine, alanine, ornithine, L-leucine,2-phenylalanine, glutamic acid, threonine, etc.; organic sugars or sugaralcohols, such as lactose, trehalose, stachyose, mannitol, sorbitol,xylitol, ribitol, myoinisitol, galactitol, glycerol and the like,including cyclitols such as inositol; polyethylene glycol; amino acidpolymers; sulfur containing reducing agents, such as urea, glutathione,thioctic acid, sodium thioglycolate, thioglycerol,alpha.-monothioglycerol and sodium thio sulfate; low molecular weightpolypeptides (i.e. <10 residues); proteins such as human serum albumin,bovine serum albumin, gelatin or immunoglobulins; hydrophylic polymers,such as polyvinylpyrrolidone monosaccharides, such as xylose, mannose,fructose, glucose; disaccharides such as lactose, maltose, sucrose andtrisaccacharides such as raffinose; and polysaccharides such as dextran.Stabilizers may be present in the range from 0.1 to 10,000 weights perpart of weight active protein.

Non-ionic surfactants or detergents (also known as “wetting agents”) maybe added to help solubilize the therapeutic agent as well as to protectthe therapeutic protein against agitation-induced aggregation, whichalso permits the formulation to be exposed to shear surface stressedwithout causing denaturation of the protein. Suitable non-ionicsurfactants include polysorbates (20, 80, etc.), polyoxamers (184, 188etc.), PLURONIC® polyols, polyoxyethylene sorbitan monoethers(TWEEN-20®, TWEEN-80®, etc.). Non-ionic surfactants may be present in arange of about 0.05 mg/ml to about 1.0 mg/ml, preferably about 0.07mg/ml to about 0.2 mg/ml.

Additional miscellaneous excipients include bulking agents, (e.g.,starch), chelating agents (e.g., EDTA), antioxidants (e.g., ascorbicacid, methionine, vitamin E), and cosolvents. The formulation herein mayalso contain more than one active compound as necessary for theparticular indication being treated, preferably those with complementaryactivities that do not adversely affect each other. For example, it maybe desirable to further provide an immunosuppressive agent. Suchmolecules are suitably present in combination in amounts that areeffective for the purpose intended. The active ingredients may also beentrapped in microcapsule prepared, for example, by coascervationtechniques or by interfacial polymerization, for example,hydroxymethylcellulose or gelatin-microcapsule andpoly-(methylmethacylate) microcapsule, respectively, in colloidal drugdelivery systems (for example, liposomes, albumin microspheres,microemulsions, nano-particles and nanocapsules) or in macroemulsions.Such techniques are disclosed in Remington's Pharmaceutical Sciences,16th edition, Osal, Ed. (1980).

The formulations to be used for in vivo administration should besterile. This is readily accomplished, for example, by filtrationthrough sterile filtration membranes. Sustained-release preparations maybe prepared. Suitable examples of sustained-release preparations includesemi-permeable matrices of solid hydrophobic polymers containing theantibody, which matrices are in the form of shaped articles, e.g.,films, or microcapsules. Examples of sustained-release matrices includepolyesters, hydrogels (for example, poly(2-hydroxyethyl-methacrylate),poly(vinylalcohol)), polylactides (U.S. Pat. No. 3,773,919), copolymersof L-glutamic acid and ethyl-L-glutamate, non-degradable ethylene-vinylacetate, degradable lactic acid-glycolic acid copolymers such as theLUPRON DEPOT™ (injectable microspheres composed of lactic acid-glycolicacid copolymer and leuprolide acetate), and poly-D-(−)-3-hydroxybutyricacid. While polymers such as ethylene-vinyl acetate and lacticacid-glycolic acid enable release of molecules for over 100 days,certain hydrogels release proteins for shorter time periods. Whenencapsulated antibodies remain in the body for a long time, they maydenature or aggregate as a result of exposure to moisture at 37° C.resulting in a loss of biological activity and possible changes inimmunogenicity. Rational strategies can be devised for stabilizationdepending on the mechanism involved. For example, if the aggregationmechanism is discovered to be intermolecular S—S bond formation throughthio-disulfide interchange, stabilization may be achieved by modifyingsulfhydryl residues, lyophilizing from acidic solutions, controllingmoisture content, using appropriate additives, and developing specificpolymer matrix compositions.

The amount of therapeutic polypeptide, antibody, or fragment thereofwhich will be effective in the treatment of a particular disorder orcondition will depend on the nature of the disorder or condition, andcan be determined by standard clinical techniques. Where possible, it isdesirable to determine the dose-response curve and the pharmaceuticalcompositions of the invention first in vitro, and then in useful animalmodel systems prior to testing in humans.

In a preferred embodiment, an aqueous solution of therapeuticpolypeptide, antibody or fragment thereof is administered bysubcutaneous injection. Each dose may range from about 0.5 μg to about50 μg per kilogram of body weight, or more preferably, from about 3 μgto about 30 μg per kilogram body weight.

The dosing schedule for subcutaneous administration may vary from once amonth to daily depending on a number of clinical factors, including thetype of disease, severity of disease, and the subject's sensitivity tothe therapeutic agent.

Therapeutic Uses of Anti-Notch-3 Antibodies

It is contemplated that the antibodies of the present invention may beused to treat a mammal. In one embodiment, the antibody is administeredto a nonhuman mammal for the purposes of obtaining preclinical data, forexample. Exemplary nonhuman mammals to be treated include nonhumanprimates, dogs, cats, rodents and other mammals in which preclinicalstudies are performed. Such mammals may be established animal models fora disease to be treated with the antibody or may be used to studytoxicity of the antibody of interest. In each of these embodiments, doseescalation studies may be performed on the mammal.

An antibody, with or without a therapeutic moiety conjugated to it,administered alone or in combination with cytotoxic factor(s) can beused as a therapeutic. The present invention is directed toantibody-based therapies which involve administering antibodies of theinvention to an animal, a mammal, or a human, for treating aNotch3-mediated disease, disorder, or condition. The animal or subjectmay be a mammal in need of a particular treatment, such as a mammalhaving been diagnosed with a particular disorder, e.g., one relating toNotch3. Antibodies directed against Notch3 are useful against cancer andother Notch3-associated diseases including neurological disorders,diabetes, rheumatoid arthritis, vascular related diseases, and Alagillesyndrome in mammals, including but not limited to cows, pigs, horses,chickens, cats, dogs, non-human primates etc., as well as humans. Forexample, by administering a therapeutically acceptable dose of ananti-Notch3 antibody, or antibodies, of the present invention, or acocktail of the present antibodies, or in combination with otherantibodies of varying sources, disease symptoms may be ameliorated orprevented in the treated mammal, particularly humans.

Therapeutic compounds of the invention include, but are not limited to,antibodies of the invention (including fragments, analogs andderivatives thereof as described herein) and nucleic acids encodingantibodies of the invention as described below (including fragments,analogs and derivatives thereof and anti-idiotypic antibodies asdescribed herein). The antibodies of the invention can be used to treat,inhibit, or prevent diseases, disorders, or conditions associated withaberrant expression and/or activity of Notch3, including, but notlimited to, any one or more of the diseases, disorders, or conditionsdescribed herein. The treatment and/or prevention of diseases,disorders, or conditions associated with aberrant expression and/oractivity of Notch3 includes, but is not limited to, alleviating at leastone symptom associated with those diseases, disorders, or conditions.Antibodies of the invention may be provided in pharmaceuticallyacceptable compositions as known in the art or as described herein.

Anti-Notch3 antibodies of the present invention may be usedtherapeutically in a variety of diseases. The present invention providesa method for preventing or treating Notch3-mediated diseases in amammal. The method comprises administering a disease preventing ortreating amount of anti-Notch3 antibody to the mammal. The anti-Notch3antibody binds to Notch3 and antagonizes its function. Notch3 signalinghas been linked to various diseases such as various cancers (Haruki, etal., Cancer Res 65:3555 (2005); Park, et al., Cancer Res 66:6312 (2006);Lu, et al., Clin Cancer Res 10:3291 (2004)); Hedvat, et al., Br JHaematol 122:728 (2003); Buchler, et al., Ann Surg 242:791 (2005));Bellavia, et al., Proc Natl Acad Sci USA 99:3788 (2002); Screpanti, etal., Trends Mol Med 9:30 (2003)); van Limpt, et al., Cancer Lett 228:59(2005)), neurological disorders (Joutel, et al., Nature 383:707 (1996)),diabetes (Anastasi, et al., J Immunol 171:4504 (2003), rheumatoidarthritis (Yabe, et al., J Orthop Sci 10:589 (2005)), vascular relateddiseases (Sweeney, et al., FASEB J 18:1421 (2004)), and Alagillesyndrome (Flynn, et al., J Pathol 204:55 (2004)). Anti-Notch3 antibodieswill also be effective to prevent the above mentioned diseases.

The amount of the antibody which will be effective in the treatment,inhibition, and prevention of a disease or disorder associated withaberrant expression and/or activity of Notch3 can be determined bystandard clinical techniques. The dosage will depend on the type ofdisease to be treated, the severity and course of the disease, whetherthe antibody is administered for preventive or therapeutic purposes,previous therapy, the patient's clinical history and response to theantibody, and the discretion of the attending physician. The antibodycan be administered in treatment regimes consistent with the disease,e.g., a single or a few doses over one to several days to ameliorate adisease state or periodic doses over an extended time to inhibit diseaseprogression and prevent disease recurrence. In addition, in vitro assaysmay optionally be employed to help identify optimal dosage ranges. Theprecise dose to be employed in the formulation will also depend on theroute of administration, and the seriousness of the disease or disorder,and should be decided according to the judgment of the practitioner andeach patient's circumstances. Effective doses may be extrapolated fromdose-response curves derived from in vitro or animal model test systems.

For antibodies, the dosage administered to a patient is typically 0.1mg/kg to 150 mg/kg of the patient's body weight. Preferably, the dosageadministered to a patient is between 0.1 mg/kg and 20 mg/kg of thepatient's body weight, more preferably 1 mg/kg to 10 mg/kg of thepatient's body weight. Generally, human antibodies have a longerhalf-life within the human body than antibodies from other species dueto the immune response to the foreign polypeptides. Thus, lower dosagesof human antibodies and less frequent administration is often possible.Further, the dosage and frequency of administration of antibodies of theinvention may be reduced by enhancing uptake and tissue penetration(e.g., into the brain) of the antibodies by modifications such as, forexample, lipidation. For repeated administrations over several days orlonger, depending on the condition, the treatment is sustained until adesired suppression of disease symptoms occurs. However, other dosageregimens may be useful. The progress of this therapy is easily monitoredby conventional techniques and assays.

The antibody composition will be formulated, dosed and administered in amanner consistent with good medical practice. Factors for considerationin this context include the particular disorder being treated, theparticular mammal being treated, the clinical condition of theindividual patient, the cause of the disorder, the site of delivery ofthe agent, the method of administration, the scheduling ofadministration, and other factors known to medical practitioners. The“therapeutically effective amount” of the antibody to be administeredwill be governed by such considerations, and is the minimum amountnecessary to prevent, ameliorate, or treat a disease or disorder. Theantibody need not be, but is optionally formulated with one or moreagents currently used to prevent or treat the disorder in question. Theeffective amount of such other agents depends on the amount of antibodypresent in the formulation, the type of disorder or treatment, and otherfactors discussed above. These are generally used in the same dosagesand with administration routes as used hereinbefore or about from 1 to99% of the heretofore employed dosages.

The antibodies of the invention may be administered alone or incombination with other types of cancer treatments including conventionalchemotherapeutic agents (paclitaxel, carboplatin, cisplatin anddoxorbicin), anti-EGFR agents (gefitinib, erlotinib and cetuximab),anti-angiogenesis agents (bevacizumab and sunitinib), as well asimmuno-modulating agents such as interferon-α and thalidomide.

In a preferred aspect, the antibody is substantially purified (e.g.,substantially free from substances that limit its effect or produceundesired side-effects).

Various delivery systems are known and can be used to administer anantibody of the present invention, including injection, e.g.,encapsulation in liposomes, microparticles, microcapsules, recombinantcells capable of expressing the compound, receptor-mediated endocytosis(see, e.g., Wu, et al., J Biol Chem 262:4429 (1987)), construction of anucleic acid as part of a retroviral or other vector, etc.

The anti-Notch3 antibody can be administered to the mammal in anyacceptable manner. Methods of introduction include but are not limitedto parenteral, subcutaneous, intraperitoneal, intrapulmonary,intranasal, epidural, inhalation, and oral routes, and if desired forimmunosuppressive treatment, intralesional administration. Parenteralinfusions include intramuscular, intradermal, intravenous,intraarterial, or intraperitoneal administration. The antibodies orcompositions may be administered by any convenient route, for example byinfusion or bolus injection, by absorption through epithelial ormucocutaneous linings (e.g., oral mucosa, rectal and intestinal mucosa,etc.) and may be administered together with other biologically activeagents. Administration can be systemic or local. In addition, it may bedesirable to introduce the therapeutic antibodies or compositions of theinvention into the central nervous system by any suitable route,including intraventricular and intrathecal injection; intraventricularinjection may be facilitated by an intraventricular catheter, forexample, attached to a reservoir, such as an Ommaya reservoir. Inaddition, the antibody is suitably administered by pulse infusion,particularly with declining doses of the antibody. Preferably the dosingis given by injections, most preferably intravenous or subcutaneousinjections, depending in part on whether the administration is brief orchronic.

Pulmonary administration can also be employed, e.g., by use of aninhaler or nebulizer, and formulation with an aerosolizing agent. Theantibody may also be administered into the lungs of a patient in theform of a dry powder composition (See e.g., U.S. Pat. No. 6,514,496).

In a specific embodiment, it may be desirable to administer thetherapeutic antibodies or compositions of the invention locally to thearea in need of treatment; this may be achieved by, for example, and notby way of limitation, local infusion, topical application, by injection,by means of a catheter, by means of a suppository, or by means of animplant, said implant being of a porous, non-porous, or gelatinousmaterial, including membranes, such as sialastic membranes, or fibers.Preferably, when administering an antibody of the invention, care mustbe taken to use materials to which the protein does not absorb.

In another embodiment, the antibody can be delivered in a vesicle, inparticular a liposome (see Langer, Science 249:1527 (1990); Treat, etal., in Liposomes in the Therapy of Infectious Disease and Cancer,Lopez-Berestein, et al., eds., pp. 353-365 (1989); Lopez-Berestein,ibid., pp. 317-27; see generally ibid.).

In yet another embodiment, the antibody can be delivered in a controlledrelease system. In one embodiment, a pump may be used (see Langer,Science 249:1527 (1990); Sefton, CRC Crit Ref Biomed Eng 14:201 (1987);Buchwald, et al., Surgery 88:507 (1980); Saudek, et al., N Engl J Med321:574 (1989)). In another embodiment, polymeric materials can be used(see Medical Applications of Controlled Release, Langer, et al., eds.,CRC Press (1974); Controlled Drug Bioavailability, Drug Product Designand Performance, Smolen, et al., eds., Wiley (1984); Ranger, et al., JMacromol Sci Rev Macromol Chem 23:61 (1983); see also Levy, et al.,Science 228:190 (1985); During, et al., Ann Neurol 25:351 (1989);Howard, et al., J Neurosurg 71:105 (1989)). In yet another embodiment, acontrolled release system can be placed in proximity of the therapeutictarget.

The present invention also provides pharmaceutical compositions. Suchcompositions comprise a therapeutically effective amount of the antibodyand a physiologically acceptable carrier. In a specific embodiment, theterm “physiologically acceptable” means approved by a regulatory agencyof the Federal or a state government or listed in the U.S. Pharmacopeiaor other generally recognized pharmacopeia for use in animals, and moreparticularly in humans. The term “carrier” refers to a diluent,adjuvant, excipient, or vehicle with which the therapeutic isadministered. Such physiological carriers can be sterile liquids, suchas water and oils, including those of petroleum, animal, vegetable, orsynthetic origin, such as peanut oil, soybean oil, mineral oil, sesameoil and the like. Water is a preferred carrier when the pharmaceuticalcomposition is administered intravenously. Saline solutions and aqueousdextrose and glycerol solutions can also be employed as liquid carriers,particularly for injectable solutions. Suitable pharmaceuticalexcipients include starch, glucose, lactose, sucrose, gelatin, malt,rice, flour, chalk, silica gel, sodium stearate, glycerol monostearate,talc, sodium chloride, dried skim milk, glycerol, propylene, glycol,water, ethanol and the like. The composition, if desired, can alsocontain minor amounts of wetting or emulsifying agents, or pH bufferingagents. These compositions can take the form of solutions, suspensions,emulsion, tablets, pills, capsules, powders, sustained-releaseformulations and the like. The composition can be formulated as asuppository, with traditional binders and carriers such astriglycerides. Oral formulation can include standard carriers such aspharmaceutical grades of mannitol, lactose, starch, magnesium stearate,sodium saccharine, cellulose, magnesium carbonate, etc. Examples ofsuitable carriers are described in “Remington's Pharmaceutical Sciences”by E. W. Martin. Such compositions will contain an effective amount ofthe antibody, preferably in purified form, together with a suitableamount of carrier so as to provide the form for proper administration tothe patient. The formulation should suit the mode of administration.

In one embodiment, the composition is formulated in accordance withroutine procedures as a pharmaceutical composition adapted forintravenous administration to human beings. Typically, compositions forintravenous administration are solutions in sterile isotonic aqueousbuffer. Where necessary, the composition may also include a solubilizingagent and a local anesthetic such as lignocaine to ease pain at the siteof the injection. Generally, the ingredients are supplied eitherseparately or mixed together in unit dosage form, for example, as a drylyophilized powder or water free concentrate in a hermetically sealedcontainer such as an ampoule or sachette indicating the quantity ofactive agent. Where the composition is to be administered by infusion,it can be dispensed with an infusion bottle containing sterilepharmaceutical grade water or saline. Where the composition isadministered by injection, an ampoule of sterile water for injection orsaline can be provided so that the ingredients may be mixed prior toadministration.

The invention also provides a pharmaceutical pack or kit comprising oneor more containers filled with one or more of the ingredients of thepharmaceutical compositions of the invention. Optionally associated withsuch container(s) can be a notice in the form prescribed by agovernmental agency regulating the manufacture, use or sale ofpharmaceuticals or biological products, which notice reflects approvalby the agency of manufacture, use or sale for human administration.

In addition, the antibodies of the present invention may be conjugatedto various effector molecules such as heterologous polypeptides, drugs,radionucleotides, or toxins. See, e.g., PCT publications WO 92/08495; WO91/14438; WO 89/12624; U.S. Pat. No. 5,314,995; and EP 396,387. Anantibody or fragment thereof may be conjugated to a therapeutic moietysuch as a cytotoxin (e.g., a cytostatic or cytocidal agent), atherapeutic agent, or a radioactive metal ion (e.g., alpha-emitters suchas, for example, 213Bi). A cytotoxin or cytotoxic agent includes anyagent that is detrimental to cells. Examples include paclitaxol,cytochalasin B, gramicidin D, ethidium bromide, emetine, mitomycin,etoposide, tenoposide, vincristine, vinblastine, colchicin, doxorubicin,daunorubicin, dihydroxy anthracin dione, mitoxantrone, mithramycin,actinomycin D, 1-dehydrotestosterone, glucocorticoids, procaine,tetracaine, lidocaine, propranolol, and puromycin and analogs orhomologues thereof. Therapeutic agents include, but are not limited to,antimetabolites (e.g., methotrexate, 6-mercaptopurine, 6-thioguanine,cytarabine, 5-fluorouracil decarbazine), alkylating agents (e.g.,mechlorethamine, thioepa chlorambucil, melphalan, carmustine (BSNU) andlomustine (CCNU), cyclothosphamide, busulfan, dibromomannitol,streptozotocin, mitomycin C, and cis-dichlorodiamine platinum (II) (DDP)cisplatin), anthracyclines (e.g., daunorubicin (formerly daunomycin) anddoxorubicin), antibiotics (e.g., dactinomycin (formerly actinomycin),bleomycin, mithramycin, and anthramycin (AMC)), and anti-mitotic agents(e.g., vincristine and vinblastine).

Techniques for conjugating such therapeutic moieties to antibodies arewell known, see, e.g., Arnon, et al., “Monoclonal Antibodies ForImmunotargeting Of Drugs In Cancer Therapy”, in Monoclonal Antibodiesand Cancer Therapy, Reisfeld, et al. (eds.), pp. 243-56 Alan R. Liss(1985); Hellstrom, et al., “Antibodies For Drug Delivery”, in ControlledDrug Delivery, 2nd ed., Robinson, et al., eds., pp. 623-53, MarcelDekker (1987); Thorpe, “Antibody Carriers Of Cytotoxic Agents In CancerTherapy: A Review,” in Monoclonal Antibodies '84: Biological AndClinical Applications, Pinchera, et al., eds., pp. 475-506 (1985);“Analysis, Results, And Future Prospective Of The Therapeutic Use OfRadiolabeled Antibody In Cancer Therapy,” in Monoclonal Antibodies ForCancer Detection and Therapy, Baldwin, et al., eds., pp. 303-16,Academic Press (1985); and Thorpe, et al., Immunol Rev 62:119 (1982).Alternatively, an antibody can be conjugated to a second antibody toform an antibody heteroconjugate. See, e.g., U.S. Pat. No. 4,676,980.

The conjugates of the invention can be used for modifying a givenbiological response, the therapeutic agent or drug moiety is not to beconstrued as limited to classical chemical therapeutic agents. Forexample, the drug moiety may be a protein or polypeptide possessing adesired biological activity. Such proteins may include, for example, atoxin such as abrin, ricin A, pseudomonas exotoxin, or diphtheria toxin;a protein such as tumor necrosis factor, α-interferon, β-interferon,nerve growth factor, platelet derived growth factor, tissue plasminogenactivator, an apoptotic agent, e.g., TNF-α, TNF-β, AIM I (See,International Publication No. WO 97/33899), AIM II (See, InternationalPublication No. WO 97/34911), Fas Ligand (Takahashi, et al., IntImmunol, 6:1567 (1994)), VEGI (See, International Publication No. WO99/23105); a thrombotic agent; an anti-angiogenic agent, e.g.,angiostatin or endostatin; or biological response modifiers such as, forexample, lymphokines, interleukin-1 (“IL-1”), interleukin-2 (“IL-2”),interleukin-6 (“IL-6”), granulocyte macrophage colony stimulating factor(“GM-CSF”), granulocyte colony stimulating factor (“G-CSF”), or othergrowth factors.

Articles of Manufacture

In another embodiment of the invention, an article of manufacturecontaining materials useful for the treatment of the disorders describedabove is provided. The article of manufacture comprises a container anda label. Suitable containers include, for example, bottles, vials,syringes, and test tubes. The containers may be formed from a variety ofmaterials such as glass or plastic. The container holds a compositionwhich is effective for preventing or treating the condition and may havea sterile access port (for example, the container may be an intravenoussolution bag or a vial having a stopper pierceable by a hypodermicinjection needle). The active agent in the composition is the antibody.The label on, or associated with, the container indicates that thecomposition is used for treating the condition of choice. The article ofmanufacture may further comprise a second container comprising apharmaceutically acceptable buffer, such as phosphate-buffered saline,Ringer's solution, and dextrose solution. It may further include othermaterials desirable from a commercial and user standpoint, includingother buffers, diluents, filters, needles, syringes, and package insertswith instructions for use.

Antibody-Based Gene Therapy

In a another aspect of the invention, nucleic acids comprising sequencesencoding antibodies or functional derivatives thereof, are administeredto treat, inhibit or prevent a disease or disorder associated withaberrant expression and/or activity of Notch3, by way of gene therapy.Gene therapy refers to therapy performed by the administration to asubject of an expressed or expressible nucleic acid. In this embodimentof the invention, the nucleic acids produce their encoded protein thatmediates a therapeutic effect. Any of the methods for gene therapyavailable can be used according to the present invention. Exemplarymethods are described below.

For general reviews of the methods of gene therapy, see Goldspiel, etal., Clinical Pharmacy 12:488 (1993); Wu, et al., Biotherapy 3:87(1991); Tolstoshev, Ann Rev Pharmacol Toxicol 32:573 (1993); Mulligan,Science 260:926 (1993); Morgan, et al., Ann Rev Biochem 62:191 (1993);May, TIBTECH 11:155 (1993).

In a one aspect, the compound comprises nucleic acid sequences encodingan antibody, said nucleic acid sequences being part of expressionvectors that express the antibody or fragments or chimeric proteins orheavy or light chains thereof in a suitable host. In particular, suchnucleic acid sequences have promoters operably linked to the antibodycoding region, said promoter being inducible or constitutive, and,optionally, tissue-specific.

In another particular embodiment, nucleic acid molecules are used inwhich the antibody coding sequences and any other desired sequences areflanked by regions that promote homologous recombination at a desiredsite in the genome, thus providing for intrachromosomal expression ofthe antibody encoding nucleic acids (Koller, et al., Proc Natl Acad SciUSA 86:8932 (1989); Zijlstra, et al., Nature 342:435 (1989)). Inspecific embodiments, the expressed antibody molecule is a single chainantibody; alternatively, the nucleic acid sequences include sequencesencoding both the heavy and light chains, or fragments thereof, of theantibody.

Delivery of the nucleic acids into a patient may be either direct, inwhich case the patient is directly exposed to the nucleic acid ornucleic acid-carrying vectors, or indirect, in which case, cells arefirst transformed with the nucleic acids in vitro, then transplantedinto the patient. These two approaches are known, respectively, as invivo or ex vivo gene therapy.

In a specific embodiment, the nucleic acid sequences are directlyadministered in vivo, where it is expressed to produce the encodedproduct. This can be accomplished by any of numerous methods known inthe art, e.g., by constructing them as part of an appropriate nucleicacid expression vector and administering it so that they becomeintracellular, e.g., by infection using defective or attenuatedretrovirals or other viral vectors (see U.S. Pat. No. 4,980,286), or bydirect injection of naked DNA, or by use of microparticle bombardment(e.g., a gene gun; Biolistic, Dupont), or coating with lipids orcell-surface receptors or transfecting agents, encapsulation inliposomes, microparticles, or microcapsules, or by administering them inlinkage to a peptide which is known to enter the nucleus, byadministering it in linkage to a ligand subject to receptor-mediatedendocytosis (see, e.g., Wu, et al., J Biol Chem 262:4429 (1987)) (whichcan be used to target cell types specifically expressing the receptors),etc. In another embodiment, nucleic acid-ligand complexes can be formedin which the ligand comprises a fusogenic viral peptide to disruptendosomes, allowing the nucleic acid to avoid lysosomal degradation. Inyet another embodiment, the nucleic acid can be targeted in vivo forcell specific uptake and expression, by targeting a specific receptor(see, e.g., PCT Publications WO 92/06180; WO 92/22635; WO92/20316;WO93/14188, WO 93/20221). Alternatively, the nucleic acid can beintroduced intracellularly and incorporated within host cell DNA forexpression, by homologous recombination (Koller, et al., Proc Natl AcadSci USA 86:8932 (1989); Zijlstra, et al., Nature 342:435 (1989)).

In a specific embodiment, viral vectors that contain nucleic acidsequences encoding an antibody of the invention are used. For example, aretroviral vector can be used (see Miller, et al., Meth Enzymol 217:581(1993)). These retroviral vectors contain the components necessary forthe correct packaging of the viral genome and integration into the hostcell DNA. The nucleic acid sequences encoding the antibody to be used ingene therapy are cloned into one or more vectors, which facilitate thedelivery of the gene into a patient. More detail about retroviralvectors can be found in Boesen, et al., Biotherapy 6:291 (1994), whichdescribes the use of a retroviral vector to deliver the mdrl gene tohematopoietic stem cells in order to make the stem cells more resistantto chemotherapy. Other references illustrating the use of retroviralvectors in gene therapy are: Clowes, et al., J Clin Invest 93:644(1994); Kiem, et al., Blood 83:1467 (1994); Salmons, et al., Human GeneTherapy 4:129 (1993); and Grossman, et al., Curr Opin Gen and Dev 3:110(1993).

Adenoviruses may also be used in the present invention. Adenoviruses areespecially attractive vehicles in the present invention for deliveringantibodies to respiratory epithelia. Adenoviruses naturally infectrespiratory epithelia. Other targets for adenovirus-based deliverysystems are liver, the central nervous system, endothelial cells, andmuscle. Adenoviruses have the advantage of being capable of infectingnon-dividing cells. Kozarsky, et al., Curr Opin Gen Dev 3:499 (1993)present a review of adenovirus-based gene therapy. Bout, et al., HumanGene Therapy 5:3 (1994) demonstrated the use of adenovirus vectors totransfer genes to the respiratory epithelia of rhesus monkeys. Otherinstances of the use of adenoviruses in gene therapy can be found inRosenfeld, et al., Science 252:431 (1991); Rosenfeld, et al., Cell68:143 (1992); Mastrangeli, et al., J Clin Invest 91:225 (1993); PCTPublication WO94/12649; Wang, et al., Gene Therapy 2:775 (1995).Adeno-associated virus (AAV) has also been proposed for use in genetherapy (Walsh, et al., Proc Soc Exp Biol Med 204:289 (1993); U.S. Pat.Nos. 5,436,146; 6,632,670; and 6,642,051).

Another approach to gene therapy involves transferring a gene to cellsin tissue culture by such methods as electroporation, lipofection,calcium phosphate mediated transfection, or viral infection. Usually,the method of transfer includes the transfer of a selectable marker tothe cells. The cells are then placed under selection to isolate thosecells that have taken up and are expressing the transferred gene. Thosecells are then delivered to a patient.

In this embodiment, the nucleic acid is introduced into a cell prior toadministration in vivo of the resulting recombinant cell. Suchintroduction can be carried out by any method known in the art,including but not limited to transfection, electroporation,microinjection, infection with a viral or bacteriophage vectorcontaining the nucleic acid sequences, cell fusion, chromosome-mediatedgene transfer, microcell-mediated gene transfer, spheroplast fusion,etc. Numerous techniques are known in the art for the introduction offoreign genes into cells (see, e.g., Loeffler, et al., Meth Enzymol217:599 (1993); Cohen, et al., Meth Enzymol 217:618 (1993); Cline,Pharmac Ther 29:69 (1985)) and may be used in accordance with thepresent invention, provided that the necessary developmental andphysiological functions of the recipient cells are not disrupted. Thetechnique should provide for the stable transfer of the nucleic acid tothe cell, so that the nucleic acid is expressible by the cell andpreferably heritable and expressible by its cell progeny.

The resulting recombinant cells can be delivered to a patient by variousmethods known in the art. Recombinant blood cells (e.g., hematopoieticstem or progenitor cells) are preferably administered intravenously. Theamount of cells envisioned for use depends on the desired effect,patient state, etc., and can be determined by one skilled in the art.

Cells into which a nucleic acid can be introduced for purposes of genetherapy encompass any desired, available cell type, and include but arenot limited to epithelial cells, endothelial cells, keratinocytes,fibroblasts, muscle cells, hepatocytes; blood cells such as Tlymphocytes, B lymphocytes, monocytes, macrophages, neutrophils,eosinophils, megakaryocytes, granulocytes; various stem or progenitorcells, in particular hematopoietic stem or progenitor cells, e.g., asobtained from bone marrow, umbilical cord blood, peripheral blood, fetalliver, etc.

In one embodiment, the cell used for gene therapy is autologous to thepatient. Nucleic acid sequences encoding an antibody of the presentinvention are introduced into the cells such that they are expressibleby the cells or their progeny, and the recombinant cells are thenadministered in vivo for therapeutic effect. In a specific embodiment,stem or progenitor cells are used. Any stem and/or progenitor cellswhich can be isolated and maintained in vitro can potentially be used inaccordance with this embodiment of the present invention (see e.g. PCTPublication WO 94/08598; Stemple, et al., Cell 71:973 (1992); Rheinwald,Meth Cell Bio 21A:229 (1980); Pittelkow, et al., Mayo Clinic Proc 61:771(1986)).

EXAMPLES Example 1 Generation of Immunogen: Notch3 ExtracellularDomain-Fc Fusion Protein

Anti-Notch3 monoclonal antibodies that specifically bind to theLIN12/dimerization domain (herein after “LD”) of human Notch3 weregenerated using a recombinant Notch3-Fc fusion protein as immunogencomprising Notch3 LD fused to a gamma 1 Fc region at the carboxyterminal end. Specifically, the immunogen comprised amino acid residues1378 to 1640 of Notch3 LD (See FIG. 1) and human γ1Fc fusion protein(Notch3 LD/Fc). A control antibody was generated comprising the Notch3EGF repeat region from amino acid residues 43 to 1377 (designated255A-79).

Notch3 protein sequence was analyzed using an internet-based researchsoftware and service (Motif Search). Human liver and pancreatic RNAs(Ambion, Inc. Austin, Tex.) were used as templates to synthesize thefirst strand of cDNA using a standard commercially available cDNAsynthesis kit. The cDNAs encoding the Notch3 LD and the EGF repeatregion were PCR-amplified in the presence of Betaine (1-2M) and DMSO(5%). The PCR-synthesized Notch3-LD DNA fragment (˜0.8 kb) andNotch3-EGF repeat DNA fragment (−4 kb) were cloned into expressionvectors comprising a Hisγ1Fc in the commercially available vector pSecor in the commercially available vector pCD3.1, each bearing a differentantibiotic marker. This cloning resulted in two expression plasmids, oneexpressing a Notch3-LD/Fc fusion protein and the other expressing aNotch3-EGF/Fc fusion protein.

To facilitate the plasmid construction and to enhance the expression ofthe various Notch 3 recombinant proteins, oligonucleotides correspondingto the leader peptide sequence comprising the first 135 base pairs ofthe Notch3 nucleic acid coding sequence were generated. Theseoligonucleotides contained some changes in the wobble coding positionsto lower the GC content. All nucleotide sequence changes were silent,i.e., no amino acid sequence changes (FIG. 14A). After annealing theoligonucleotides together, the engineered leader peptide coding sequencewas linked to the rest of the coding sequence by PCR-SOE (Ho, et al.,Gene 77:51 (1989); Horton, et al., Bio Techniques 8:528 (1990)) (SeeFIG. 15). This leader peptide coding sequence was used in Notch3-LD/Fcand Notch3 expression constructs. Therefore, both of the Fc fusionproteins comprise a signal peptide linked to the N-terminus, and a humanγ1Fc sequence fused to the C-terminus. The amino acid sequence ofNotch3-LD, including the leader peptide, is shown in FIG. 14 and SEQ IDNO:6.

Expression of Notch3-EGF/Fc and Notch3-LD/Fc fusion proteins wasverified by transient transfection of the Notch3 expression plasmidsinto 293T (ATCC Number CRL-11268, Manassas, Va.) and CHO cells(Invitrogen, Carlsbad, Calif.), respectively. Prior to transfection,cells were cultured in DMEM (Invitrogen, Carlsbad, Calif.) growth mediumcontaining 10% fetal calf serum (FCS), 2 mM of glutamine, and 1×essential amino acid solution followed by seeding about 3-5×10⁵ cellsper well in 6-well plate and growing for approximately 24 hours. Threemicrograms each of the Notch3 fusion protein expression plasmids weretransfected into cells in each well using a LIPOFECTAMINE™ 2000transfection system (Invitrogen, Carlsbad, Calif.) following themanufacturer's protocol. After transfection, the cells were cultured infresh growth medium and cultured in a CO₂ incubator for approximately40-48 hours before subjecting to Notch3 fusion protein expressionanalysis. Alternatively, after transfection, the cells were cultured ingrowth medium for 3-4 hours, then switched to DMEM medium containing 2%FCS and cultured for approximately 60-66 hours before drawingconditioned medium for secreted protein analysis.

Stable cell lines were generated for both Notch3-LD/Fc (His-Fcγ/pSecvector) and Notch3-EGF/Fc (His-Fcγ/pSec vector). Each plasmid wastransfected into CHO cells. After transfection, the cells were culturedin DMEM growth medium overnight, then switched to growth medium with 800μg/ml hygromycin and cultured at least two weeks until the cells notcarrying Notch3 expression plasmid were eliminated by the antibiotics.Conditioned media from the stable cell lines were subjected to Westernblot analysis.

Stable or transient transfected cells were assayed for expression andsecretion of Notch3-LD/Fc or Notch3-EGF/Fc fusion protein. Transfectedcells harvested from culture dishes were washed once with phosphatebuffered saline (PBS) and resuspended in deionized water, mixed with anequal volume of 2× protein sample loading buffer (BioRad, Hercules,Calif.) and then heated at about 100° C. for 10 minutes. Secretedprotein was analyzed using conditioned medium mixed with an equal volumeof 2× protein sample loading buffer and heated at 100° C. for 10minutes. The samples were separated using 4-15% gradient SDS-PAGE. Theproteins were transferred from the gel to a PVDF membrane (BioRad,Hercules, Calif.), which was blocked in 5% non-fat dry milk in PBST (PBSwith 0.05% TWEEN-20®) for at least one hour prior to transfer ofprotein.

Notch3-EGF/Fc and Notch3-LD/Fc fusion proteins were detected byincubating with γFc-specific, HRP-conjugated antibody (Sigma, St Louis,Mo.) in blocking buffer for one hour at room temperature. The membranewas washed three times in PBST and developed with a chemiluminescentsubstrate.

For Notch3 domain/Fc fusion protein purification, CHO stable cell linesas described above were cultured in DMEM with 2% FCS for up to 5 days.One liter of conditioned medium was collected and subjected to protein-Abead-packed column chromatography for affinity binding. The column waswashed with PBS, and the bound proteins were eluted in 50 mM citratebuffer (pH 2.8), and the pH was brought to neutral by adding 1 MTris-HCl buffer (pH 8). Purity of the protein was assessed by proteingel analysis using 4-15% gradient SDS-PAGE. Protein concentration wasassayed using Coomassie blue reagent following the manufacturer'sprotocol (Pierce, Rockford, Ill.). Through this procedure, milligramquantities of Notch3-LD/Fc and Notch3-EGF/Fc protein were purified forimmunization and ELISA binding assays.

Example 2 Generation of Anti-Notch3 MAbs

Male A/J mice (Harlan, Houston, Tex.), 8-12 weeks old, were injectedsubcutaneously with 25 μg of Notch3-EGF/Fc or Notch3-LD/Fc in completeFreund's adjuvant (Difco Laboratories, Detroit, Mich.) in 200 μl of PBS.Two weeks after the injections and three days prior to sacrifice, themice were again injected intraperitoneally with 25 μg of the sameantigen in PBS. For each fusion, single cell suspensions were preparedfrom spleen of an immunized mouse and used for fusion with Sp2/0 myelomacells; 5×10⁸ of Sp2/0 and 5×10⁸ of spleen cells were fused in a mediumcontaining 50% polyethylene glycol (M.W. 1450) (Kodak, Rochester, N.Y.)and 5% dimethylsulfoxide (Sigma, St. Louis, Mo.). The cells were thenadjusted to a concentration of 1.5×10⁵ spleen cells per 200 μl of thesuspension in Iscove medium (Invitrogen, Carlsbad, Calif.), supplementedwith 10% fetal bovine serum, 100 units/ml of penicillin, 100 μg/ml ofstreptomycin, 0.1 μM hypoxanthine, 0.4 μM aminopterin, and 16 μMthymidine. Two hundred microliters of the cell suspension were added toeach well of about sixty 96-well plates. After around ten days, culturesupernatants were withdrawn for screening their antibody-bindingactivity using ELISA.

The 96-well flat bottom IMMULON® II microtest plates (DynatechLaboratories, Chantilly, Va.) were coated using 100 μl of Notch3-EGF/Fcor Notch3-LD/Fc (0.1 μg/ml) in (PBS) containing 1× Phenol Red and 3-4drops pHix/liter (Pierce, Rockford, Ill.) and incubated overnight atroom temperature. After the coating solution was removed by flicking ofthe plate, 200 μl of blocking buffer containing 2% BSA in PBSTcontaining 0.1% merthiolate was added to each well for one hour to blocknon-specific binding. The wells were then washed with PBST. Fiftymicroliters of culture supernatant from each fusion well were collectedand mixed with 50 μl of blocking buffer and then added to the individualwells of the microtiter plates. After one hour of incubation, the wellswere washed with PBST. The bound murine antibodies were then detected byreaction with horseradish peroxidase (HRP)-conjugated, Fc-specific goatanti-mouse IgG (Jackson ImmunoResearch Laboratories, West Grove, Pa.).HRP substrate solution containing 0.1% 3,3,5,5-tetramethyl benzidine and0.0003% hydrogen peroxide was added to the wells for color developmentfor 30 minutes. The reaction was terminated by the addition of 50 ml of2 M H₂SO₄/well. The OD at 450 nm was read with an ELISA plate reader(Molecular Devices, Sunnyvale, Calif.).

Among 185 hybridomas isolated and analyzed, two hybridoma clones frommice immunized with Notch3-LD/Fc generated Notch3 antagonizingantibodies, which were further characterized. The ELISA usingsupernatant from the two hybridoma clones producing MAbs 256A-4 and256A-8 showed strong binding activity to the purified Notch3 LD/FCfusion protein to which it was generated and did not bind to humanNotch1-LD/Fc (LIN/dimerization domain fused to Fc region at the carboxylterminus) or a control human Fc protein (data not shown) (Table 1).Later studies using functional assays also demonstrated that MAbs 256A-4and 256A-8 specifically antagonize Notch3 relative to Notch1 and Notch2(data not shown).

TABLE 1 ELISA OD readings of anti-Notch3 Mabs using hybridomasupernatant Notch3-LD/Fc Notch1-LD/Fc Mean S.D. Mean S.D. 256A-4 4.0000.000 0.106 0.004 256A-8 4.000 0.000 0.115 0.014 Control IgG1* 0.0640.006 0.066 0.006 *Control IgG was an irrelevant IgG1 monoclonalantibody.

The positive hybridoma clones from this primary ELISA screening werefurther isolated by single colony-picking and a second ELISA assay asdescribed above was done to verify specific binding to the chosenimmunogen. The confirmed hybridoma clones were expanded in larger scalecultures. The monoclonal antibodies (MAbs) were purified from the mediumof these large scale cultures using a protein A affinity column. Theanti-Notch3 MAbs were then characterized using cell-based bindingassays, microscopy, Western blot, and FACS analysis.

Example 3 Cell-Based Binding Assays for Anti-Notch3 MAbs

The cell-based binding assays used to characterize the anti-Notch3 MAbsrequired cloning a full-length human Notch3 open reading frame into avector, in this case PCDNA™ 3.1/Hygro (Invitrogen, Carlsbad, Calif.).The Notch3-coding region was synthesized by RT-PCR using human livertumor RNA (Ambion, Inc., Austin, Tex.) as a template. The final plasmidconstruct, Notch3/Hygro, expressed a full-length Notch3 protein asdepicted in FIG. 1. A stable cell line expressing Notch3 was generatedby transfection of Notch3/Hygro plasmid construct into 293T cells (ATCCNo. CRL-11268) using a LIPOFECTAMINE™ 2000 kit following the sameprocedure as described in Example 1. After transfection, the cells werecultured in DMEM growth medium overnight, then reseeded in growth mediumwith 200 μg/ml hygromycin and cultured for 12-14 days. Well-isolatedsingle colonies were picked and grown in separate wells until enoughclonal cells were amplified. Stable 293T clones that were resistant tohygromycin selection and expressed high levels of Notch3 protein wereidentified by Western blot analysis, and by fluorescentelectromicroscopy using polyclonal anti-Notch3 antibodies (R&D Systems,Minneapolis, Minn.).

A partial Notch3 expression plasmid containing only the NotchLIN12/dimerization (LD) domain and the transmembrane (TM) domain wasalso constructed by PCR and subcloning into PCDNA™ 3.1 vector(Invitrogen, Carlsbad, Calif.). This plasmid construct also contains aV5 tag at its C-terminus and was termed Notch3-LDTM/V5. A stable cellline expressing this plasmid, Notch3-LDTM/V5, was generated according tothe procedure described in Example 1.

Human Sup-T1 cell line (ATCC No. CRL-1942) naturally expressing Notch3was also confirmed by Western blot. Sup-T1 cells were grown in RPMI1640media containing 10% fetal calf serum, 2 mM of glutamine and 1×essential amino acid solution.

Cell-based antibody-binding was assessed using FMAT™ (fluorescencemacro-confocal high-throughput screening) 8100 HTS System (AppliedBiosystems, Foster City, Calif.) following the protocol provided by themanufacturer. Cell lines naturally expressing Notch3 or stablytransfected with Notch3 expression constructs were seeded in 96-wellplates. Alternatively, transiently transfected 293T or CHO cells wereseeded in the 96-well plate. The cells were seeded at a density of30,000-50,000 cells per well. After 20-24 hours, anti-Notch3 MAbs and1×PBS reaction buffer were added to the wells and incubated for one hourat 37° C. Cy-5-conjugated anti-mouse IgG antibody was added in the wellsafter removal of primary antibodies.

Cell-based antibody-binding was also assessed by fluorescence-activatedcell sorter (FACS) using an internally generated 293T/Notch3-stable cellline and two cancer lines, human Sup-T1 and A2780 cell lines (UK ECACCNo. Cat. No. 93112519), which both naturally express Notch3 (data notshown). Cells were first incubated with anti-Notch3 MAbs in 1×PBS. Afterthree washes, the cells were incubated with fluorescentmolecule-conjugated secondary antibody. The cells were resuspended,fixed in 1×PBS with 0.1% paraformaldehyde, and analyzed by FACS (BDSciences, Palo Alto, Calif.). The results indicated that both MAbs bindto Notch3 receptor expressed either from recombinant plasmid constructsor as native protein in cultured cells (Table 2). However, Western blotshowed that when the Notch3 receptor or the Notch3-LD/Fc fusion proteinare denatured in SDS-PAGE and transferred to nylon blot membrane, theanti-Notch3 MAbs no longer bind, suggesting a conformational epitope.Transiently transfected 293T cells containing a Notch3/Hygro plasmidwere also stained with immunofluorescence as described above andobserved by fluorescent microscopy.

TABLE 2 Binding activity of anti-Notch3 MAbs in cell-based FACS analysisshown as mean fluorescent intensity Monoclonal Antibody293T/Notch3-stable cell line Sup-T1 256A-4 195 43 256A-8 189 45 negativecontrol* 21 23 positive control** 198 74

The cell-based FMAT™ and FACS analyses confirmed that both MAbs 256A-4and 256A-8 indeed bind to the Notch3 receptor expressed either fromrecombinant plasmid constructs or as native protein in cultured cells(Table 2 and Table 3).

TABLE 3 Summary of anti-Notch3 MAbs binding activity in cell-basedFMAT ™ mAb 256A-4 mAb 256A-8 mAb G3 Notch3 (full-length)/ + + − 293T

G3 is a negative control human IgG1 Mab. A positive binding signal wasdetermined based on the FMAT™ signal read-out that was significantlyhigher than G3 and other negative hybridoma clones (p>0.01). Thenegative signal of G3 FMAT™ binding read-out was considered background.Transiently transfected 293T cells with Notch3/Hygro plasmid were alsostained with immunofluorescence as described above and observed byfluorescent microscopy.

Example 4 Western Blot Analysis of Anti-Notch3 MAb Binding Activity

Western blot was performed to assess the anti-Notch3 MAbs' bindingactivity to Notch3 under denaturing conditions, as well as expressionlevels of Notch3 and other Notch-related proteins in human cell lines.Purified Notch3-LD/Fc fusion protein was combined with protein loadingbuffer. Protein samples were also prepared from the transiently orstably transfected cells described in Example 1, which were harvestedfrom culture dishes, washed once with PBS, resuspended in total cellularprotein extract buffer (Pierce, Rockford, Ill.), and heated at 100° C.for 10 minutes after adding equal volume of 2× protein sample loadingbuffer. All samples were separated by electrophoresis in a 4-15%gradient SDS-PAGE. The proteins were transferred from gel to PVDFmembrane and anti-Notch3 MAbs were applied to the Western blot membraneas the primary detection antibody. An HRP-conjugated secondary antibodywas used for detection and the signal generated using a chemiluminescentsubstrate as described above. Positive control antibodies against humanFc, V5 tag, Notch3 and Notch1 were purchased from Invitrogen, R&DSystems, Santa Cruz Biotechnologies, and Orbigen.

Western blot analysis showed that MAbs 256A-4 and 256A-8 do not bind toNotch3-LD/Fc under denaturing conditions, which is in distinct contrastto the results observed in ELISA and FACS analyses where Notch3LIN12/heterodimerization domains are maintained in native molecularconformation. Therefore, it is concluded that MAbs 256A-4 and 256A-8bind to multiple epitopes in Notch3-LD that have to be maintained intheir native conformation. This conclusion was confirmed by the resultsfrom epitope mapping discussed in Example 8 below.

Example 5 Assessing Functionality of Anti-Notch3 MAbs by LuciferaseReporter Assay

A. Plasmid Constructs

The full length Notch3 expression construct described in Example 3 abovewas confirmed by sequencing, and is identical to the published sequencedepicted in FIG. 1. Human Jagged1 plasmid was obtained from OriGene(Rockville, Md.), and verified by sequencing as identical toNM_(—)000214 (NCBI/GENBANK® accession number). Because the OriGeneJagged1 plasmid did not have an antibiotic selection marker, the Not Ifragment containing Jagged1 coding sequence was transferred into PCDNA™3.1/Hygromycin. A 3.7 Kb subclone of human Jagged2 cDNA was generated byfirst strand cDNA synthesis from human T-cell leukemia cell line, HH(ATCC No. CRL-2105) and PCR-amplified. The Jagged2 cDNA was subsequentlysubcloned. The expression of Notch3, Jagged1, and Jagged2 was verifiedby transient transfection and Western blot as described in Example 4.

To generate a luciferase reporter plasmid for Notch signaling, twocomplementary oligonucleotide primers containing tandem repeats of CBF1binding motif were synthesized having the following sequences:

(SEQ ID NO 7) 5′GCTCGAGCTCGTGGGAAAATACCGTGGGAAAATGAACCGTGGGAAAATCTCGTGG  (SEQ ID NO 8) 5′GCTCGAGATTTTCCCACGAGATTTTCCCACGGTTC

These two oligoprimers were annealed at 65° C. in 100 mM of NaCl witheach oligo at a concentration of 4 mM. After annealing to each other,the primers were extended by PCR. The PCR product was cloned into acommercially available vector. The insert was verified by sequencing,which contains four tandem repeats of CBF1 binding motif and twoflanking Xho I sites. The insert was excised using Xho I and ligateddownstream of the firefly luciferase reporter coding sequence. Afterluciferase reporter assay and sequencing analysis, plasmid clones witheight repeats of CBF1 binding motifs were selected and designatedCBF1-Luc.

B. Stable Cell Line Generation

Two stable cell lines were generated for functional assays using humanembryonic kidney cell lines (HEK293). One cell line contained theNotch3-expressing plasmid and CBF1-Luc reporter plasmid integrated intothe nuclear genome. This cell line was generated by cotransfectingNotch3/hygromycin and CBF1-Luc plasmids into 293T cells usingLIPOFECTAMINE™ 2000 transfection system according to the manufacturer'sprotocol. Stable transfection cell clones were selected against 200μg/ml hygromycin in DMEM growth medium, and screened by luciferasereporter assay and Western blot. A cell line with relatively high levelof Notch3 expression (based on Western blot) and luciferase activity wasselected for use in functional assay, and designated NC85.

The second stable cell line contained a Notch ligand expressionconstruct, such as Jagged1 or Jagged2, or PCDNA™ 3.1 as negativecontrol. Stable cell lines expressing human Jagged1 or harboring PCDNA™3.1 were generated by transfection into 293T cells and selection againsthygromycin as described above. Jagged2 was subcloned, transfected into a293T cell line and expected to be integrated into a specific locus inthe genome. Hygromycin-resistant cells were selected as above.

C. Luciferase Reporter Assay Under Coculture Conditions

NC85 cells were mixed and cocultured with another 293T cell line stablyexpressing human Jagged1 (Jagged1/293T), Jagged2/293F, or PCDNA™3.1/293T, respectively, for 24 to 48 hours. At the end of theco-culture, the media was removed by aspiration, cells were lysed in 1×Passive Lysis Buffer (E1501, Promega, Madison, Wis.) and luciferaseactivities were assayed using the Luciferase Assay System followingmanufacturer's protocol (E1501, Promega, Madison, Wis.) in TD-20/20luminometer (Turner Designs Instrument, Sunnyvale, Calif.). Asillustrated in FIG. 6 and FIG. 7, when NC85 cells were cocultured withJagged1/293T or with Jagged2/293F, the luciferase activity was increased2-4 fold as compared to that of coculturing with PCDNA™ 3.1/293T cells.To assess the inhibitory effect of anti-Notch3 MAbs, the antibodies wereadded to the cell culture at beginning of seeding and mixing ofcocultured cells. (256-A, 256A-8 and an EGF-Repeat Domain control255A-79).

D. Luciferase Reporter Assay by Culturing Cells on Notch Ligand-CoatedPlates

Regular 96-well tissue culture plates from Becton Dickinson Labware(#18779, Palo Alto, Calif.) were coated with rat Jagged1/Fc, human DLL-4(R&D Systems, Minneapolis, Minn.) or Human Fc (Jackson ImmunoResearch,West Grove, Pa.), bovine serum albumin (Sigma, St Louis, Mo.). Onehundred microliters of each protein (3 μg/ml in PBS) was distributed ina well and maintained at room temperature or 4° C. for at least 8 hoursuntil the coating solution was removed before use. NC85 cells or cancercells were seeded at 3-5×10⁴ cells per well and allowed to grow for28-48 hours. The luciferase reporter assay and antibody inhibition assaywere performed as described in Section C above. The luciferase reporterassay demonstrated the two MAbs 256A-4 and 256A-8 binding toLIN12/dimerization domain almost completely blocked Jagged1 andJagged2-induced luciferase reporter activity (FIGS. 6 and 7). Incontrast, a MAb specifically binding to Notch3-EGF domain (255A-79), asa control, only inhibited Jagged1-induced luciferase reporter activity(about 60% inhibition, FIG. 6), but not Jagged2-induced luciferasereporter activity (FIG. 7). The ability of MAbs 256A-4 and 256A-8 toblock DLL-4-induced luciferase reporter activity is shown in FIG. 8.

Additional functional assays demonstrated that MAbs 256A-4 and 256A-8inhibited ligand-induced up-regulation of Notch target genes. 293T cellsexpressing recombinant Notch3 were cultured on Jagged-1-coated plates.In the presence of MAbs 256A-4 and 256A-8, up-regulation of HESS andHEY2, two Notch target genes, was inhibited, as measured by quantitativeRT-PCR (data not shown).

To verify whether the anti-Notch3 MAbs can bind to native Notch3expressed in human cancer cells and block the receptor signaling, areporter assay was performed using two ovarian cancer cell lines,OV/CAR3 and A2780. Both 256A-4 and 256A-8 significantly blockedJagged1-induced Notch signaling mediated by native Notch3 in OV/CAR3cells (FIG. 9 a). Similarly, both MAbs inhibited about 50% of luciferaseactivity induced by D114 coated on the plate (FIG. 9 b). The latterresult is consistent with the fact that both Notch1 and Notch3 areexpressed in A2780 cells. These results suggest that the anti-Notch3MAbs can inhibit native Notch3-mediated signaling in cancer cells.

Example 6 Apoptosis Assay

Annexin V is an early apoptotic marker on the cell surface, and theapoptotic cell population can be marked by fluorophore-labeledanti-Annexin V antibody and quantified by FACS analysis. NC85 cells wereseeded at 5-6×10⁴ cells per well in Fc- or Jagged1/Fc-coated 96-wellplate as described above and maintained in serum-free DMEM medium for 24hours. Apoptotic cells were stained by FITC-labeled anti-Annexin Vantibody (BD Biosciences, Palo Alto, Calif.) and analyzed by FACS. Cellscultured on Jagged1/Fc-coated surface had significantly lower apoptoticcell population comparing to those cultured on Fc-coated plate (FIG.10). To study the antibody's functional effect, anti-Notch3 MAbs wereadded in cell culture at the beginning of the study. As shown in FIG.10, anti-Notch3 MAbs 256A-4 and 256A-8 blocked about 50-65% of the cellsurvival effect induced by Jagged1.

Example 7 Cell Migration Assays, Invasion Assays, and Morphology Assays

In vitro cell migration and invasion assays are frequently used toassess metastasis potential of cancer cells. These assays were performedto assay the inhibitory effect exerted by the anti-Notch3 MAbs on thetumorgenic 293T/Notch3-stable cell line (NC85). The invasion assay wasperformed using COSTAR® 48-well insert plate (Sigma-Aldrich, St. Louis,Mo.). The insert divides the well into upper and lower chambers whichare separated by a porous membrane (pore diameter=8 μm) at the bottom ofthe insert. Notch ligands, Jagged1/Fc, DLL-4, or human Fc, wereimmobilized on the membrane surface as describe in above sections. NC85cells were seeded at 100,000 cells per well and maintained in serum-freeDMEM in the upper chamber and 10% FCS/DMEM in the lower chamber. After10-24 hours, cells that remained on the top surface of the insertmembrane were removed, and the cells that passed the membrane adheringon the bottom of the insert membrane were stained by 0.05% crystalvelvet in PBS. The dye was extracted from the cells by 30% acetic acidand absorption readings at 590 nm were recorded. The anti-Notch3 MAbswere added to cell culture 24 hours before seeding NC85 cells in theCOSTAR® assay plate and all MAbs were added to the cell culture 24 hoursbefore seeding NC85 cells in the COSTAR® assay plate. Fresh MAbs wereadded to maintain the same concentration in the migration assay plate.Experimental results are shown in FIG. 11A.

The invasion assay was performed using Becton Dickinson 48-wellMATRIGEL™ plate (BD Labware, Palo Alto, Calif.). The cell culture wellwas divided by an insert well into upper and lower chambers, which areseparated by a porous membrane (pore diameter=8 μm) at the bottom of theinsert well. An optimized density of MATRIGEL™ matrix was coated on themembrane top surface and fibronectin was coated on the membrane bottomsurface by the manufacturer. NC85, Jagged1/293T, and pcDNA3.1/293T cellswere mixed pair-wise such as indicated in FIG. 11B. A total of 6-10×10⁴cells were seeded in each well in the 48-well MATRIGEL™ plate andcultured in growth medium for 24 hours. The cells that remained on topof the insert membrane in the upper chamber were removed and the cellsthat passed the membrane adhering on the bottom of the insert membranewere stained by 0.05% crystal velvet in PBS. The dye was extracted andabsorption measurements were as described in the previous section. MAbswere added at the beginning of the mixed cell culture. The results areshown in FIG. 11B.

The cell migration assay results showed that when NC85 cells werecultured on Jagged1-coated membrane, the activation of Notch3 signalingsignificantly increased cell migration, and MAbs 256A-4 and 256A-8clearly inhibited the migration (FIG. 11A). The invasion experimentshowed a similar trend (FIG. 11B).

Additionally, the effect of MAbs 256A-4 and 256A-8 on Jagged-1-inducedformation of cell “spheres” was examined. When 293T cellsover-expressing Notch3 were cultured on Jagged-1-coated plates, thecells formed loosely attached “cell balls” or “spheres.” In the presenceof MAbs 256A-4 and 256A-8, however, formation of these cell spheres wasinhibited (data not shown).

Example 8 Mapping the Binding Epitope of Anti-Notch3 MAbs

A. Domain Swap Strategy and Rationale

First, the antagonist Notch3 MAbs bind to Notch3 LIN12/dimerizationdomain (LD), but not to the homologous human Notch1 LIN12/dimerizationdomain (See FIGS. 12 and 13). Second, the anti-Notch3 MAbs do not bindto denatured Notch3 protein in Western blot as discussed in Example 4,indicating the MAbs bind to conformational epitopes. Third, Notch3 andNotch1 share approximately 55% amino acid sequence homology in theLIN12/dimerization domain, and therefore it was concluded that a domainswap between Notch3 and Notch1 within this region would not disrupt theprotein conformation.

B. Generating Domain Swap Fusion Protein Constructs

Sequence analysis indicated that Notch3 has three LIN12 repeats and itsdimerization domain is divided into two segments. Therefore, five domainswap protein constructs were generated with each of the three LIN12repeats and the two dimerization segments replaced by the correspondingdomains of Notch1. The domain swap constructs were generated usingPCR-SOE (Ho, et al., Gene 77:51 (1989); Horton, et al., BioTechniques8:528 (1990)) as illustrated in FIG. 12. PCR and PCR-SOE reactions wereperformed using PCR with 1M Betaine and 5% DMSO added to the reaction.PCR thermocycling was almost same for PCR and PCR-SOE except that theannealing step of each PCR cycle was extended one minute in PCR-SOE. Thefinal PCR-SOE product was subcloned and verified by sequencing. Theplasmid clone with the correct insert sequence was cleaved with Nhe Iand Xho I to excise the insert, which was gel-purified and subcloned.The five Notch3/Notch1 domain swap constructs are illustrated in FIG.12. To facilitate the epitope mapping, the human IgG kappa chainsignaling peptide was used as leader peptide in the domain swapconstructs. The amino acid sequences are shown in FIGS. 16 and 17.

Notch1-LD cDNA was PCR-amplified using PCR and methods described in theabove section. The first strand cDNA template was synthesized from PA-1cell total RNA (ATCC No. CRL-1572). The human IgG kappa chain leaderpeptide coding sequence was PCR-amplified, used as leader peptide tolink to the 5′ of Notch1-LD by PCR-SOE and subcloned in His-γ1Fc/pSec.

Based on ELISA analysis results, target domains L1, D1 and D2 werefurther divided into subdomains. ELISA binding analysis using thesubdomain expression constructs showed that only L1 and D2 were requiredfor the Notch3 MAb binding. The D1 domain was not required. Therefore,L1 and D2 domains were divided into clusters of amino acid mutations forfurther analysis of the specific binding site. Constructs containing L1and D2 subdomain swap or clusters of amino acid mutations as shown inFIG. 16 and FIG. 17 were generated.

C. Expression of Notch3/Notch1 Domain Swap Fusion Protein

Notch3/Notch1-LD domain swap plasmids were transiently transfected inCHO cells using LIPOFECTAMINE™ 2000 transfection system. CHO cells wereseeded in DMEM growth medium with 10% FCS at 0.8-1×10⁶ cells per well inE-well plate, maintained in CO₂ incubator overnight before transfection.The cells were recovered after transfection in the growth medium forabout 3 hours, then switched to DMEM with 2% FCS, and cultured for threedays. The conditioned media were harvested and centrifuged at 3500 rpmfor 10 minutes. The supernatant containing Notch3-LD domain swap proteinsecreted from CHO was collected and prepared for Western blot and ELISAbinding analyses. ELISA showed that all the domain-swap fusion proteinswere expressed and secreted in conditioned medium (Table 4), which wasfurther confirmed by Western blot analysis (data not shown).

The ELISA readings used anti-human Fc antibody as detection antibodyshowing all the proteins were expressed in conditioned medium. HumanIgG/Fc was used as a control. The starting point of human IgG/Fc coatedin each well is 100 ng.

TABLE 4 ELISA Readings Dilution N1-LD N3-LD L1-swap L2-swap L3-swapD1-swap D2-swap hIgG-Fc Statistics: mean 1 3.2000 3.3445 3.4380 3.09703.2910 3.2870 3.4110 3.5510 0.250000 3.1305 2.7625 2.9890 2.7390 2.90503.0225 2.9570 3.4995 0.062500 2.3785 1.3870 2.8145 1.2835 2.6855 2.25752.3240 3.5805 0.015625 1.0085 0.3960 1.5245 0.3865 1.7350 0.9110 0.88003.2355 0.003906 0.3300 0.1075 0.4755 0.1220 0.5970 0.3450 0.2130 1.85850.000977 0.2095 0.0400 0.1640 0.1105 0.1780 0.1635 0.0615 0.58650.000244 0.1340 0.0225 0.0500 0.0595 0.0575 0.1045 0.0275 0.14456.104E−05 0.1000 0.0135 0.0405 0.0505 0.0230 0.0575 0.0305 0.03151.526E−05 0.0975 0.0165 0.0205 0.0430 0.0180 0.0400 0.0155 0.02203.815E−06 0.0580 0.0140 0.0135 0.0300 0.0150 0.0425 0.0235 0.02309.537E−07 0.0540 0.0125 0.0155 0.0245 0.0215 0.0480 0.0145 0.01652.384E−07 0.0415 0.0125 0.0145 0.0305 0.0155 0.0370 0.0150 0.0190Statistics: S.D. 1 0.0778 0.0290 0.0679 0.0255 0.0933 0.1018 0.02830.0071 0.250000 0.0191 0.0304 0.0354 0.0396 0.0693 0.1619 0.1202 0.01480.062500 0.0898 0.0919 0.0007 0.1096 0.0318 0.0021 0.0071 0.02900.015625 0.0474 0.0354 0.0106 0.0417 0.1075 0.0071 0.0325 0.14500.003906 0.0523 0.0177 0.0460 0.0113 0.0453 0.0339 0.0057 0.05730.000977 0.0092 0.0057 0.0042 0.0191 0.0156 0.0205 0.0007 0.09550.000244 0.0226 0.0092 0.0014 0.0106 0.0064 0.0035 0.0049 0.02766.104E−05 0.0113 0.0007 0.0064 0.0035 0.0057 0.0134 0.0064 0.00641.526E−05 0.0021 0.0035 0.0049 0.0042 0.0000 0.0028 0.0007 0.00283.815E−06 0.0113 0.0028 0.0021 0.0000 0.0042 0.0064 0.0007 0.00579.537E−07 0.0014 0.0007 0.0007 0.0007 0.0064 0.0057 0.0021 0.00782.384E−07 0.0120 0.0035 0.0049 0.0021 0.0007 0.0113 0.0014 0.0127

Abbreviations for proteins used in the ELISA binding assays of Table 4include: N1-LD, Notch1-LD/Fc. N3-LD, Notch3-LD/Fc. L1-swap: 1st LIN12domain swap. L2-swap: 2nd LIN12 domain swap. L3-swap: 3rd LIN12 domainswap. D1-swap: 1st dimerization domain swap. D2-swap: 2nd dimerizationdomain swap. hIgG-Fc, human IgG Fc.

D. Epitope Binding Analysis Using ELISA

The 96-well flat bottom IMMULON II microtest plates (DynatechLaboratories, Chantilly, Va.) were coated with anti-human Fc antibody(Jackson ImmunoResearch) by adding 100 μl of the antibody (0.1 μg/ml) inphosphate buffered saline (PBS) containing 1× Phenol Red and 3-4 dropspHix/liter (Pierce, Rockford, Ill.), and incubated overnight at roomtemperature. After the coating solution was removed by flicking of theplate, 200 μl of blocking buffer containing 2% BSA in PBST and 0.1%merthiolate was added to each well for one hour to block non-specificbinding. The wells were then washed with PBST. Fifty microliters of theabove conditioned medium from each transfection of Notch3/Notch1 domainswap construct were collected, mixed with 50 μl of blocking buffer, andadded to the individual wells of the microtiter plates. After one hourof incubation, the Notch3/Notch1-LD domain swap protein was captured bythe coated anti-Fc antibody, and the wells were washed with PBST.Anti-Notch3 MAbs and isotype-matched control MAbs were serially dilutedin blocking buffer as above, and 50 μl of the diluted MAbs were added ineach well to assess binding to the bound Notch3/Notch1 domain swapprotein. Horseradish peroxidase (HRP)-conjugated, Fc-specific goatanti-mouse IgG was used for detection. HRP substrate solution containing0.1% 3,3,5,5-tetramethyl benzidine and 0.0003% hydrogen peroxide wasadded to the wells for color development for 30 minutes. The reactionwas terminated by addition of 50 ml of 2 M H2SO₄/well. The OD at 450 nmwas read with an ELISA reader. Subdomain swap constructs and clusters ofmutations were similarly examined by ELISA analysis above.

ELISA binding experiments using MAbs 256A-4 and 256A-8 against thedomain-swap proteins showed that the swap of the 1st LIN12 domain (L1)and 2nd dimerization domain (D2) completely abolished all the three MAbsbinding, while the swap of 1st dimerization domain (D1) abolishedbinding of MAbs 256A-4 and 256A-8 (FIG. 13 B&C). Swap of the 3rd LIN12domain (L3) significantly weakened the binding. Nevertheless, both MAbswere still able to bind to the fusion protein. The swap of the 2nd LIN12domain had no interference with the binding of the MAbs (FIGS. 13B andC). A positive control antibody, which was previously mapped to bind tothe 1st LIN12 domain, bound to all domain swap fusion protein except L1(FIG. 13D). In contrast, isotype control negative antibody, G3, does notbind to any of the domain swap fusion proteins in the ELISA assay (datanot shown). It was concluded from the above experiments that the 1stLIN12 domain and 2nd dimerization domain were required for MAbs 256A-4and 256A-8 binding.

To further map the epitopes in the 1st LIN12 domain (L1) to whichanti-Notch3 MAbs bind, the L1 domain was further divided into threesubdomains, L1-sub1, L1-sub2 and L1-sub3, and swapped with thecorresponding sequences in Notch1 (FIG. 16). An ELISA binding assayshowed that L1-sub1 swap has no inhibitory effects on binding activity,and L1-sub2 and L1-sub3 swap abolished binding (FIG. 16). In L1-sub2 andL1-sub3 regions, there are five clusters of amino acid residues thatdiffer between Notch3 and Notch1. Therefore, swap fusion proteinconstructs were generated within these five clusters of amino acids(FIG. 16). ELISA analysis demonstrated that L1-cluster4 swap had noinhibition on all three MAbs binding. The remaining four clusters ofswap partially or completely abolished the anti-Notch MAbs binding.Thus, those four clusters of amino acid residues represented fourdifferent epitopes to which the MAbs bind. L1-cluster3 (amino acids:DRE) and L1-cluster5 (amino acids: SVG) are required. L1-cluster1 (aminoacids: AKR) and cluster2 (amino acids: DQR) also played a role inanti-Notch3 MAb binding, whose mutations significantly weakened the MAbbinding.

To map the epitopes in the 2^(nd) dimerization (D2) domain of Notch3 towhich anti-Notch3 MAbs bind, the D2 domain was further divided into fivesubdomains, D2-sub1, D2-sub2, D2-sub3, D2-sub4 and D2-sub5. Thesequences in those subdomains were swapped with the correspondingsequences in Notch1 (FIG. 17). An ELISA binding assay showed that MAbs256A-4 and 256A-8 have strong binding to D1-sub2 and D2-sub3 swap, butnot to D2-sub1 and D2-sub4 swap. Both MAbs showed weak binding toD2-sub5 (FIG. 17). Therefore, the data suggested that D2-sub1 andD2-sub4 are required for the anti-Notch3 MAb binding and D2-sub5 mayhelp the binding activity.

Both MAbs 256A-4 and 256A-8 are antagonistic antibodies binding to theconformational epitope comprising L1 and D2, while another antibody256A-13 that binds only to L1 is agonistic (See co-pending U.S.application Ser. No. 11/874,682, filed Oct. 18, 2007). Furthermore,agonistic 256A-13 competes with antagonistic 256A-4 for an epitopewithin L1, and the epitope mapping studies suggest that they bind to anoverlapping epitope on L1. The major difference is that the antagonisticantibodies also bind to D2, while the agonistic antibody does not. Totest the hypothesis that simultaneous binding to L1 and D2 isresponsible for the antagonistic activity, an antibody, 256A-2 bindingto a similar epitope in D2 as 256A-4 was analyzed. MAb 256A-2 is neitherantagonistic nor agonistic (data not shown). Studies showed that 256A-2does not compete with 256A-13 and can bind to Notch3 simultaneously.Furthermore, 256A-2 and 256A-13 individually can partially compete with256A-4, however, in combination these two antibodies completely blockbinding of 256A-4 to Notch3 (data not shown). Studies also showed thatseparate binding of two antibodies to the epitopes in L1 and D2 does notlead to the inhibition of ligand-dependent Notch3 activation, suggestingthat the antagonistic antibodies form a bridge, possibly locking andstabilizing the L1 and D2 interaction, and preventing the ligand inducedconformational changes. (See FIG. 18)

Example 9 Sequencing of Anti-Notch3 MAbs

Because antibody binding properties are dependent on the variableregions of both heavy chain and light chain, the variable sequences of256A-4 and 256A-8 were subtyped and sequenced. The antibody IgG subtypewas determined using an ISOSTRIP™ mouse monoclonal antibody isotypingkit (Roche Diagnostics, Indianapolis, Ind.). The results showed thatboth MAbs, 256A-4 and 256A-8 have an IgG₁ heavy chain and a kappa lightchain.

The variable region sequences of heavy chain and light chain weredecoded through RT-PCR and cDNA cloning. Total RNAs from hybridomaclones 256A-4 and 256A-8 were isolated using an RNeasy Mini kitfollowing the manufacturer's protocol (QIAGEN, Valencia, Calif.). Thefirst strand cDNA was synthesized using the RNA template andSUPERSCRIPT® III reverse transcriptase kit. The variable region of lightchain and heavy chain cDNAs were PCR-amplified from the first strandcDNA using degenerative forward primers covering the 5′-end of mousekappa chain coding region and a reverse primer matching the constantregion at the juncture to the 3′-end of the variable region, or usingdegenerative forward primers covering the 5′-end of mouse heavy chaincoding region and a constant region reverse primer in mouse heavy chain.The PCR product was cloned into a commercially available vector andsequenced by Lone Star Lab (Houston, Tex.). The nucleotide sequenceswere analyzed utilizing the DNASTAR® computer software program (DNASTAR,Inc., Madison, Wis.). Each anti-Notch3 MAb sequence was determined bysequences from multiple PCR clones derived from the same hybridomaclone.

MAb 256A-4 contains 123 and 116 amino acid residues, respectively, inits variable region of heavy chain and light chain (FIGS. 4A and 4B).MAb 256A-8 consists of 122 and 123 amino acid residues in heavy chainand light chain variable regions, respectively (FIGS. 5A and 5B).

Example 10 Impact of Notch3 Antagonistic Antibodies on MetalloproteaseCleavage of Notch3

Notch receptor activation involves ligand induced metalloproteasecleavage at juxtamembrane site (S2) generating an extracellular subunit.This cleavage is an essential prerequisite to S3 cleavage to release theactivated Notch intracellular region. Both 256A-4 and 256A-8 were foundto require the presence of at least a portion of the Notch3 L1 and D2domains for their bindings. These two domains are not located in closeproximity in the linear sequence, but rather are on two separatepolypeptides, suggesting these antibodies may stabilize an inactive,autoinhibited Notch configuration. To test whether the antagonizingantibodies can inhibit sequential Notch activation events, including twoproteolytic cleavages, 293T cells stably expressing a recombinant Notch3receptor (NC85 cells) are treated with either immobilized recombinantJagged-1 or cocultured with 293T cells expressing Jagged-1. The solubleextracellular subunits generated by proteolytic cleavage in the culturemedium are detected by an ELISA assay using an antibody bound to a solidsurface that recognizes the Notch3 cleavage product. Notch3 antagonisticMAbs are expected to decrease the generation of soluble Notch3extracellular subunits in the conditioned medium, whereas non-functionalNotch3 binding antibodies would not.

To directly detect the S2 cleavage fragment, an 7.5% SDS PAGEelectrophoresis and Western blot with Notch3 C-terminal antibody areperformed. The S2 fragment is 57 amino acids residues smaller andmigrates slightly faster than the non-cleaved Notch3 small subunit(transmembrane subunit).

To examine whether Notch3 antagonistic MAbs inhibit ligand-inducedmetalloprotease cleavage of Notch3 at S2, 293T cells expressingrecombinant Notch3 were treated with the γsecretase inhibitor compound E(1 μM) for 4 hours, which stabilizes the product of cleavage at site S2,allowing it to accumulate. In the presence of MAbs 256A-4 and 256A-8,Jagged-1-induced metalloprotease cleavage of Notch3 at S2 was inhibited(data not shown).

Example 11 Efficacy Study Using Human Cancer Models in Xenograft Mice

A. Human Cancer Cells and Tumorigenic Cells

Human cancer cell lines with Notch3 expression such as HCC2429, HCC95may be obtained from Academic Institutes, or from the ATCC. The293T/PCDNA™ 3.1, and 293T/Notch3 (NC85) cells are generated bytransfecting 293T with related genes and selecting with hygromycin asdescribe in previous sections. All cells are cultured in DMEM or RPMI1640 medium with 10% fetal bovine serum, sodium pyruvate, nonessentialamino acids, L-glutamine, vitamin solution, and penicillin-streptomycin(Flow Laboratories, Rockville, Md.). Cell lines are incubated in amixture of 5% CO₂ and 95% air at 37° C. in an incubator. Cultures aremaintained for no longer than 3 weeks after recovery from frozen stocks.Logarithmically growing single-cell suspensions cells with ≧90%viability are used for tumor cells injection after washing with PBS.

B. Animals

Mice are obtained from, for example, the Animal Production Area of theNational Cancer Institute at Frederick Cancer Research and DevelopmentCenter, Frederick, Md. The animals are purpose-bred and areexperimentally naïve at the outset of the study. Mice selected for usein the studies are chosen to be as uniform in age and weight aspossible. They are 6-8 weeks of age and their body weights at initiationof weight range from approximately 18 to 25 grams. Records of the datesof birth for the animals used in this study are retained in the studyraw data, and the weight range at the time of group assignment isspecified in the report. Each animal is identified by a numbered eartag. The animals are group housed by treatment group (4 mice/cage) inpolystyrene disposable shoe-box cages containing cellulose bedding,meeting or exceeding NIH guidelines. During the course of the study, theenvironmental conditions in the animal room is monitored and maintainedwithin a temperature range of 18-26° C., and the relative humidity isrecorded daily. A 12-hour light/dark illumination cycle is maintainedthroughout the study. Animals have irradiated food. No contaminants areknown to be present in the food at levels that would interfere with theresults of this study. Autoclaved water is available to each animal viawater bottles. No contaminants are known to be present in the water atlevels that would interfere with the results of this study. Prior toassignment to the study, all study animals are acclimatized to theirdesignated housing for at least 7 days prior to the first day of dosing.

C. Tumor Models and Efficacy Studies

Mice are anesthetized using sodium pentobarbital (50 mg/kg body weight)and placed in the right lateral decubitus position. Cancer cells, suchas non-small cell lung cancer (NSCLC) cell lines, HCC2429 (Haruki, etal. Cancer Res. 65:3555 (2005)), HCC95 (From Dr. John Mina), and H2122(ATCC No. CRL5985), in 50 μl Hank's containing 10% MATRIGEL™ matrix areinjected into the left lobe of the lungs. After the tumor-cellinjection, the mice are turned to the left lateral decubitus positionand observed for 45-60 min until they recover fully. Records of tumorcell injections are maintained in the raw study data.

All animals are observed within their cages at least once daily duringstudy and clinical findings recorded in the study raw data. Animals thatshow pronounced detrimental effects may be removed from the study shouldit be deemed necessary. Body weight is measured once each week duringthe treatment. Cancer tissues from each mouse, where available, areharvested and stored for potential future biological characterization.

Example 12 Assay for Notch3 Related Diseases

To identify other Notch3 related diseases, one can sequence the Notch3gene from patient samples, perform FISH (fluorescence in situhybridization) and CGH (comparative genomic hybridization) analysis tolook for translocation and gene amplification using patient cells, orperform immunohistochemistry to check for the over-expression of Notch3receptor using patient tissue or tumor sections. In addition, one canisolate and culture cells from a patient suspected of having a Notch3associated disease and study the impact of an antagonistic antibody ofthe present invention on cell migration, invasion, survival andproliferation. Protocols for cell migration and invasion assay aredescribed in Example 7 and the protocol for an apoptosis assay isdescribed in Example 6. For the cell proliferation assay, cells culturedfrom patient samples are be seeded in 96-well plate coated with andwithout Notch ligands. Antagonistic antibodies are added at thebeginning of the culture. Cell numbers are counted at specific timepoints using trypan blue staining. Notch3 FISH and CGH analysis may beperformed using the published protocols of Park, et al. (Cancer Res, 66:12 (2006)).

Those skilled in the art will recognize, or be able to ascertain usingno more than routine experimentation, many equivalents to the specificembodiments of the invention described herein. Such equivalents areintended to be encompassed by the following claims.

1. An isolated polypeptide comprising the amino acid sequence of the variable heavy (“VH”) chain region of a monoclonal antibody that specifically binds to Notch3, wherein the antibody specifically binds to a conformational epitope of a Notch3 fragment consisting of amino acids 1378-1640 of SEQ ID NO:1, and wherein the antibody inhibits Notch3 signaling.
 2. The polypeptide of claim 1, wherein the antibody binds to amino acid residues in the LIN12 domain (SEQ ID NO:9) and the dimerization domain (SEQ ID NO:18).
 3. The polypeptide of claim 1, wherein the VH chain region comprises CDR-H1 of SEQ ID NO:32, CDR-H2 of SEQ ID NO:33, and CDR-H3 of SEQ ID NO:34.
 4. The polypeptide of claim 3, wherein the VH chain region comprises SEQ ID NO:2.
 5. The polypeptide of claim 1, wherein the VH chain region comprises CDR-H1 of SEQ ID NO:38, CDR-H2 of SEQ ID NO:39, and CDR-H3 of SEQ ID NO:40.
 6. The polypeptide of claim 5, wherein the VH chain region comprises SEQ ID NO:4.
 7. An isolated polypeptide comprising the amino acid sequence of the variable light (“VL”) chain region of a monoclonal antibody that specifically binds to Notch3, wherein the antibody specifically binds to a conformational epitope of a Notch3 fragment consisting of amino acids 1378-1640 of SEQ ID NO:1, and wherein the antibody inhibits Notch3 signaling.
 8. The polypeptide of claim 7, wherein the antibody binds to amino acid residues in the LIN12 domain (SEQ ID NO:9) and the dimerization domain (SEQ ID NO:18).
 9. The polypeptide of claim 7, wherein the VL chain region comprises CDR-L1 of SEQ ID NO:35, CDR-L2 of SEQ ID NO:36, and CDR-L3 of SEQ ID NO:37.
 10. The polypeptide of claim 9, wherein the VL chain region comprises SEQ ID NO:3.
 11. The polypeptide of claim 7, wherein the VL chain region comprises CDR-L1 of SEQ ID NO:41, CDR-L2 of SEQ ID NO:42, and CDR-L3 of SEQ ID NO:43.
 12. The polypeptide of claim 11, wherein the VL chain region comprises SEQ ID NO:5.
 13. An isolated nucleic acid encoding a polypeptide comprising the amino acid sequence of the VH chain region of a monoclonal antibody that specifically binds to Notch3, wherein the antibody specifically binds to a conformational epitope of a Notch3 fragment consisting of amino acids 1378-1640 of SEQ ID NO:1, and wherein the antibody inhibits Notch3 signaling.
 14. The nucleic acid of claim 13, wherein the antibody binds to amino acid residues in the LIN12 domain (SEQ ID NO:9) and the dimerization domain (SEQ ID NO:18).
 15. The nucleic acid of claim 13, wherein the VH chain region comprises CDR-H1 of SEQ ID NO:32, CDR-H2 of SEQ ID NO:33, and CDR-H3 of SEQ ID NO:34.
 16. The nucleic acid of claim 15, wherein the VH chain region comprises SEQ ID NO:2.
 17. The nucleic acid of claim 13, wherein the VH chain region comprises CDR-H1 of SEQ ID NO:38, CDR-H2 of SEQ ID NO:39, and CDR-H3 of SEQ ID NO:40.
 18. The nucleic acid of claim 17, wherein the VH chain region comprises SEQ ID NO:4.
 19. A cell comprising the nucleic acid of claim
 13. 20. A method for producing a polypeptide comprising culturing the cell of claim 19 under conditions appropriate for production of a polypeptide and isolating the polypeptide produced.
 21. An isolated nucleic acid encoding a polypeptide comprising the amino acid sequence of the VL chain region of a monoclonal antibody that specifically binds to Notch3, wherein the antibody specifically binds to a conformational epitope of a Notch3 fragment consisting of amino acids 1378-1640 of SEQ ID NO:1, and wherein the antibody inhibits Notch3 signaling.
 22. The nucleic acid of claim 21, wherein the antibody binds to amino acid residues in the LIN12 domain (SEQ ID NO:9) and the dimerization domain (SEQ ID NO:18).
 23. The nucleic acid of claim 21, wherein the VL chain region comprises CDR-L1 of SEQ ID NO:35, CDR-L2 of SEQ ID NO:36, and CDR-L3 of SEQ ID NO:37.
 24. The nucleic acid of claim 23, wherein the VL chain region comprises SEQ ID NO:3.
 25. The nucleic acid of claim 21, wherein the VL chain region comprises CDR-L1 of SEQ ID NO:41, CDR-L2 of SEQ ID NO:42, and CDR-L3 of SEQ ID NO:43.
 26. The nucleic acid of claim 25, wherein the VL chain region comprises SEQ ID NO:5.
 27. A cell comprising the nucleic acid of claim
 21. 28. A method for producing a polypeptide comprising culturing the cell of claim 27 under conditions appropriate for production of a polypeptide and isolating the polypeptide produced.
 29. An isolated nucleic acid encoding a monoclonal antibody that specifically binds to Notch3, wherein the antibody specifically binds to a conformational epitope of a Notch3 fragment consisting of amino acids 1378-1640 of SEQ ID NO:1, and wherein the antibody inhibits Notch3 signaling.
 30. The nucleic acid of claim 29, wherein the antibody binds to amino acid residues in the LIN12 domain (SEQ ID NO:9) and the dimerization domain (SEQ ID NO:18).
 31. The nucleic acid of claim 29, wherein the antibody comprises a VH chain region comprising CDR-H1 of SEQ ID NO:32, CDR-H2 of SEQ ID NO:33, and CDR-H3 of SEQ ID NO:34, and a VL chain region comprising CDR-L1 of SEQ ID NO:35, CDR-L2 of SEQ ID NO:36, and CDR-L3 of SEQ ID NO:37.
 32. The nucleic acid of claim 31, wherein the VH chain region comprises SEQ ID NO:2 and the VL chain region comprises SEQ ID NO:3.
 33. The nucleic acid of claim 29, wherein the antibody is a humanized form of a monoclonal antibody comprising the VH chain region of SEQ ID NO:2 and the VL chain region of SEQ ID NO:3.
 34. The nucleic acid of claim 29, wherein the antibody comprises a VH chain region comprising CDR-H1 of SEQ ID NO:38; CDR-H2 of SEQ ID NO:39, and CDR-H3 of SEQ ID NO:40, and a VL chain region comprising CDR-L1 of SEQ ID NO:41; CDR-L2 of SEQ ID NO:42, and CDR-L3 of SEQ ID NO:43.
 35. The nucleic acid of claim 34, wherein the VH chain region comprises SEQ ID NO:4 and the VL chain region comprises SEQ ID NO:5.
 36. The nucleic acid of claim 29, wherein the antibody is a humanized form of a monoclonal antibody comprising the VH chain region of SEQ ID NO:4 and the VL chain region of SEQ ID NO:5.
 37. A method of treating a Notch 3 related disease or disorder comprising administering to a mammal a monoclonal antibody that specifically binds to Notch3, wherein the antibody specifically binds to a conformational epitope of a Notch3 fragment consisting of amino acids 1378-1640 of SEQ ID NO:1, and wherein the antibody inhibits Notch3 signaling.
 38. The method of claim 37, wherein the antibody binds to amino acid residues in the LIN12 domain (SEQ ID NO:9) and the dimerization domain (SEQ ID NO:18).
 39. The method of claim 37, wherein the antibody comprises a VH chain region comprising CDR-H1 of SEQ ID NO:32, CDR-H2 of SEQ ID NO:33, and CDR-H3 of SEQ ID NO:34, and a VL chain region comprising CDR-L1 of SEQ ID NO:35, CDR-L2 of SEQ ID NO:36, and CDR-L3 of SEQ ID NO:37.
 40. The method of claim 39, wherein the VH chain region comprises SEQ ID NO:2, and the VL chain region comprises SEQ ID NO:3.
 41. The method of claim 37, wherein the antibody is a humanized form of a monoclonal antibody comprising the VH chain region of SEQ ID NO:2 and the VL chain region of SEQ ID NO:3.
 42. The method of claim 37, wherein the antibody comprises a VH chain region comprising CDR-H1 of SEQ ID NO:38, CDR-H2 of SEQ ID NO:39, and CDR-H3 of SEQ ID NO:40, and a VL chain region comprising CDR-L1 of SEQ ID NO:41, CDR-L2 of SEQ ID NO:42, and CDR-L3 of SEQ ID NO:43.
 43. The method of claim 42, wherein the VH chain region comprises SEQ ID NO:4, and the VL chain region comprises SEQ ID NO:5.
 44. The method of claim 37, wherein the antibody is a humanized form of a monoclonal antibody comprising the VH chain region of SEQ ID NO:4 and the VL chain region of SEQ ID NO:5.
 45. The method of claim 37, wherein the disease is T-cell acute lymphoblastic leukemia, lymphoma, liver disease involving aberrant vascularization, diabetes, ovarian cancer, diseases involving vascular cell fate, rheumatoid arthritis, pancreatic cancer, non-small cell lung cancer, plasma cell neoplasms (such as multiple myeloma, plasma cell leukemia, and extramedullary plasmacytoma), and neuroblastoma. 